1. Tetrameric Assembly of K+ Channels Requires ER-Located Chaperone Proteins 
Kai Li, Qiang Jiang, Xue Bai, Yi-Feng Yang, Mei-Yu Ruan, Shi-Qing Cai 
Molecular Cell 
Volume 65, Issue 1, Pages (January 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 65, 52-65DOI: (10.1016/j.molcel.2016.10.027)
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3. Figure 1
Genetic Screening in C. elegans Identifies DNJ-1 as an UNC-103 Channel Biogenesis Regulator
(A) Cloning of dnj-1. The red line indicates the mutated site in the third exon of the dnj-1 gene.
(B) Expression of UNC-103 proteins in unc-103A334T (control), dnj-1(lf);unc-103A334T, and dnj-1;dnj-1(lf);unc-103A334T worms (obtained by expressing wild-type dnj-1 with its own promoter in dnj-1(lf);unc-103A334T worms). White arrows indicate head neurons, and blue arrows indicate vulval muscles. Scale bars, 20 μm.
(C) Western blot analysis of UNC-103 proteins.
(D) Head thrashing (left) and egg-laying (right) behaviors in worms. n ≥ 30 worms per genotype.
(E) Fluorescent microscopy images were taken from wild-type N2 worms expressing Pdnj-1::gfp and Punc-103::mCherry gene reporters. Scale bars, 15 μm.
(F) Sequence alignment of DNJ-1 and its human homologs DNAJB12 and DNAJB14. Identical amino acids are highlighted in yellow. The mutant allele (yfh1) in DNJ-1 is shown by the red arrow. Transmembrane (TM), G/F motif, and J-domain are underlined.
All experiments were performed at least in triplicates. All data shown are mean ± SEM. ∗∗∗p < (t test). See also Figures S1–S3.
Molecular Cell  , 52-65DOI: ( /j.molcel )
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4. Figure 2
DNAJB12 and DNAJB14 Regulate the Biogenesis of Endogenous hERG Proteins
(A) Western blots (left) and densitometric quantification (right) of hERG proteins in SH-SY5Y cells. The efficiency and specificity of dnajb12 and dnajb14 siRNAs are shown in Tables S1 and S2. Effects of dnajb12 and dnajb14 siRNAs on cell viability are shown in Table S3.
(B) Representative whole-cell K+ currents recorded in SH-SY5Y cells (n = 5) before and after exposure to 1 μM E-4031 in the bath solution. The voltage protocol is shown in the lower right. Current traces at the time course between two dashed lines are shown.
(C) Representative traces (left) and current density (right) of endogenous whole-cell K+ currents recorded in SH-SY5Y cells treated with control siRNA (n = 30 cells) or siRNAs targeting dnajb12/dnajb14 (n = 36 cells).
(D) Western blot analysis of hERG proteins in hiPSC-CMs.
(E) Representative recordings of ventricular-like and atrial-like APs in hiPSC-CMs treated with control siRNA or dnajb12/dnajb14 siRNAs.
(F) APD90 of ventricular-like and atrial-like APs.
(G) Development of EAD in an hiPSC-CM treated with siRNAs against dnajb12/dnajb14.
All experiments were performed at least in triplicates. All data shown are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < (t test). See also Figure S4.
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5. Figure 3 hERG Associates with DNAJB12 and DNAJB14 in the ER
(A) Co-localization of DNAJB12/DNAJB14 and Calnexin in HEK293T cells. Calnexin and DNAJB12/DNAJB14 were detected using anti-Calnexin and anti-c-Myc antibodies, respectively. Scale bars, 10 μm.
(B) Localization of DNAJB12/DNAJB14 and hERG expressed in HEK293T cells. hERG and DNAJB12/DNAJB14 were detected using anti-HA and anti-c-Myc antibodies, respectively. Scale bars, 10 μm.
(C) CoIP of hERG with DNAJB12 or DNAJB14 expressed in HEK293T cells, and the sensitivity of the immunoprecipitated hERG proteins to Endo H.
(D) Left: schematic representations of truncated hERG proteins used in coIP experiments. The region between two red dashed lines represents the domain responsible for the interaction with DNAJB12/DNAJB14. Right: expression of truncated hERG proteins (lysate) and coIP with control (mock-transfected, lane 1), c-Myc tagged DNAJB12 (lane 2), and c-Myc tagged DNAJB14 (lane 3) are shown. Proteins were expressed in HEK293T cells and immunoprecipitated with anti-c-Myc antibody.
(E) Top: schematic representations of truncated DNAJB12 or DNAJB14 proteins used for coIP experiments. The region between two red dashed lines represents the domain responsible for interacting with hERG. Bottom: expression of truncated DNAJB12 or DNAJB14 proteins (lysate) and their interaction with hERG (coIP) are shown. All experiments were performed at least in triplicates.
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6. Figure 4
DNAJB12 and DNAJB14 Stabilize and Assemble the Nascent Channel Subunits
(A) Deactivating inward K+ current density of hERG channels expressed in HEK293T cells treated with control (n = 30 cells) or dnajb12/dnajb14 siRNAs (n = 33 cells).
(B) Effects of dnajb12 and dnajb14 siRNA treatments on the amount of hERG proteins expressed in HEK293T cells treated with vehicle (DMSO) or 10 μM MG132.
(C) Densitometric quantification of total hERG proteins in cells treated with DMSO or 10 μM MG132.
(D) Representative images of SNAP-tag labeling and fluorescence-based pulse chase of hERG proteins.
(E) Time-dependent changes in amounts of fully glycosylated, core-glycosylated (left), and the ratio of fully glycosylated to total hERG proteins (right).
(F) Western blots (left) and densitometric quantification (right) of wild-type and hERGQ725X proteins expressed in HEK293T cells treated with control or dnajb12/dnajb14 siRNAs.
(G and H) Effects of DNAJB12 (G) and DNAJB14 (H) on the oligomeric status of hERG and UNC-103 proteins expressed in a wheat germ cell-free extract with liposomes. Monomeric and tetrameric proteins were separated by native PAGE and are indicated by blue and red arrows, respectively.
All experiments were performed at least in triplicates. All data shown are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01 (t test). See also Figure S5.
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7. Figure 5
DNAJB12 and DNAJB14 Modulate the Biogenesis of Other K+ Channels
(A and B) Western blots (A) and densitometric quantification (B) of hERG, Kv4.2, EGL-36, rat Kv2.1, Kir6.2/Sur1, and Nav1.5 channels expressed in HEK293T cells after treatment with control siRNA or dnajb12/dnajb14 siRNAs. The proteins were visualized by anti-HA or anti-Nav1.5 antibodies.
(C and D) Effects of DNAJB12 on the oligomeric status of Kv4.2 (C) or rat Kv2.1 (D) proteins expressed in cell-free translation systems. Monomeric and tetrameric proteins are indicated by blue and red arrows, respectively.
All experiments were performed at least in triplicates. All data shown are mean ± SEM. ∗p < 0.05 (t test).
Molecular Cell  , 52-65DOI: ( /j.molcel )
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8. Figure 6 Oligomerization of ERG Subunits Requires Oligomerized DNAJB12
(A) Native-PAGE analysis of the oligomeric status of c-Myc-tagged DNAJB12 proteins expressed in HEK293T cells.
(B) Interaction between HA-tagged wild-type DNAJB12 and c-Myc-tagged DNAJB12Δ(G/F).
(C) Native-PAGE analyses of the oligomeric status of c-Myc-tagged DNAJB12Δ(G/F) proteins expressed in HEK293T cells.
(D) Native-PAGE analyses of the oligomeric status of hERG proteins upon co-expression with DNAJB12Δ(G/F) or DNAJB12QPD in the cell-free translation system.
(E) Expression of UNC-103 proteins in unc-103A334T (control), dnj-1(lf);unc-103A334T, dnajb12;dnj-1(lf);unc-103A334T, and dnajb12Δ(g/f);dnj-1(lf);unc-103A334T worms. The latter two strains were obtained by expressing wild-type dnajb12 and dnajb12Δ(g/f) under the dnj-1 promoter in dnj-1(lf);unc-103A334T worms, respectively. Scale bar, 20 μm.
(F) Western blot analysis of UNC-103 proteins in worms. UNC-103 proteins were visualized by anti-GFP antibody.
(G) Head thrashing (left) and egg-laying (right) behaviors in worms. At least 30 worms per genotype were tested.
(H) Native-PAGE analysis of oligomeric status of UNC-103 proteins in worms.
(I) Expression of UNC-103 proteins in dnj-1(lf);unc-103A334T (control), dnajb12;dnj-1(lf);unc-103A334T, and dnajb12qpd;dnj-1(lf);unc-103A334T worms. Scale bar, 10 μm.
(J) Western blot analyses of DNAJB12 (left) and UNC-103 (right) proteins expressed in transgenic worms.
(A, C, D, and H) Monomer, dimer, and tetramer are indicated by blue, black, and red arrows, respectively.
All experiments were performed at least in triplicates. All data shown are mean ± SEM. ∗∗p < 0.01, ∗∗∗p < (t test). See also Figure S6.
Molecular Cell  , 52-65DOI: ( /j.molcel )
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9. Figure 7
Overexpression of DNAJB14 Corrects Defective Protein Trafficking of hERG Mutants
(A) Effects of DNAJB12 or DNAJB14 overexpression on protein trafficking of hERG mutants expressed in HEK293T cells.
(B and C) Changes in deactivating inward K+ current density of wild-type hERG, hERGT65P (B), and hERGC64Y (C) upon co-expression with DNAJB14.
(D and E) Time-dependent changes in protein amounts of hERGT65P (D) and hERGC64Y (E) upon co-expression with DNAJB14 in HEK293T cells treated with 60 μg/ml cycloheximide (CHX).
(F and G) Quantitative analysis of hERGT65P (F) and hERGC64Y (G) protein levels after CHX treatment.
(H) Changes in trypsin sensitivity of hERGT65P and hERGC64Y proteins upon co-expression with DNAJB14 in HEK293T cells.
(I and J) Densitometric analysis of hERGT65P (I) and hERGC64Y (J) proteins after treatment with 0–10 μg/ml trypsin.
(K) A proposed model illustrates oligomerization of ERG subunits. After translation from the ribosome, nascent ERG peptides are bound by ER-located J-proteins, which then recruit HSP70 proteins to stabilize and fold ERG monomers. A J-protein dimer binds two ERG monomers to facilitate formation of an ERG dimer. Subsequent dimerization of J-protein dimers promotes tetrameric assembly of ERG channel subunits.
All experiments were performed at least in triplicates. All data shown are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < (t test). See also Figure S7.
Molecular Cell  , 52-65DOI: ( /j.molcel )
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