1. Cytokine-Induced GAPDH Sulfhydration Affects PSD95 Degradation and Memory 
Sajad Mir, Tanusree Sen, Nilkantha Sen 
Molecular Cell 
Volume 56, Issue 6, Pages (December 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 786-795DOI: (10.1016/j.molcel.2014.10.019)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
IL-1β Impairs Motor Coordination and Learning along with the Degradation of PSD95
(A–D) Motor coordination was measured in both cbs+/+ and cbs+/− mice after treatment with IL-1β. Number of falls/min (A) and latency to first fall (B) were measured in both groups of mice. In this test, latency to find platform (C) and time spent in the right quadrant (D) were measured for both groups of mice.
(E) Western blot hybridization to detect PSD95 in the IL-1β-injected cbs+/+ mice.
(F) Confocal microscopy of PSD95 after IL-1β (10 ng) treatment.
(G) Analysis of the number (a), density (b), and size (c) of PSD95 after IL-1β treatment.
(H and I) Western blot analysis (H) and confocal microscopy (I) of PSD95 after IL-1β treatment following depletion of CBS.
(J) Analysis of the number (a), density (b), and size (c) of PSD95 after IL-1β treatment after depletion of CBS.
(K) Depletion of PSD95 in cortex was confirmed by confocal microscopy.
(L–O) Motor coordination was measured in both cbs+/+ and cbs+/− mice after depletion of PSD95. Number of falls/min (L) and latency to first fall (M) were measured in both groups of mice. Spatial learning abilities were measured by Morris water maze (MWM) tests in both groups of mice. In this test, latency to find platform (N) and time spent in the right quadrant (O) were measured for both groups of mice. ∗p < 0.01, n = 5, mean ± SEM, one-way ANOVA. Please see Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
IL-1β-Induced Increase in Intracellular H2S Leads to the PSD95 Degradation
(A) Western blot hybridization to detect CBS protein level after treatment with increasing concentrations of IL-1β (0, 2, 5, 10 ng) into cbs+/+ mice.
(B–D) Treatment with IL-1R RNAi abolished the CBS protein level demonstrated by confocal microscopy (B), western blot analysis (C), and semiquantitative RT-PCR analysis for CBS mRNA (D).
(E) ChIP assay was used to study the binding of Sp1 to CBS promoter in cells treated with IL-1R RNAi.
(F) In primary neurons, treatment with Sp1 RNAi decreases the CBS protein after IL-1β treatment.
(G) Measurement of green fluorescent intensity of WSP1 in cells treated with CBS RNAi. Depletion of CBS level was confirmed by western blot hybridization.
(H) Measurement of PSD95 level after treatment with homocysteine (hcy) by confocal microscopy.
(I) Analysis of PSD95 puncta showed significant loss in the number (a), density (b), and size (c) after hcy treatment.
(J) Western blot analysis of PSD95 after hcy treatment following depletion of CBS.
(K) Measurement of PSD95 level after treatment with GYY4137 by confocal microscopy.
(L) Analysis of the number (a), density (b), and size (c) of PSD95 after GYY4137 treatment.
(M) Measurement of PSD95 level after treatment with GYY4137 by confocal microscopy in cells treated with either control RNAi or CBS RNAi.
(N) Analysis of the number (a), density (b), and size (c) of PSD95 after GYY4137 treatment.
(O) Western blot analysis of PSD95 after GYY4137 treatment following depletion of CBS. ∗p < 0.01, n = 10–12, mean ± SEM, Student’s t test. Please see Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
GAPDH Sulfhydration Upregulates Proteosomal Degradation of PSD95 in a Siah-Dependent Manner
(A) Maleimide assay to detect GAPDH sulfhydration in both control RNAi and CBS RNAi-treated cells following IL-1β (10 ng, 6–8 hr) treatment.
(B) Addition of GYY4137 leads to the sulfhydration of wild-type GAPDH, but not GAPDH-C150S (detected by maleimide assay).
(C) CoIP assay to detect interaction between Siah and GAPDH in CBS-depleted cells after treatment with IL-1β.
(D) Western blot analysis to measure protein level of Siah in both control and CBS RNAi-treated cells following IL-1β treatment.
(E) IP assay to detect interaction between Siah and GAPDH in cells overexpressed with either GAPDH or GAPDH-C150S after treatment with IL-1β.
(F) IP assay to detect interaction between Siah and PSD95 in CBS-depleted cells after treatment with IL-1β.
(G) Confocal microscopic analysis to study the Siah-PSD95 interaction in the IL-1β-treated CBS-depleted neurons. The arrowheads indicate the colocalization of Siah and PSD95.
(H) Measurement of Siah and CBS protein levels in both control RNAi and CBS RNAi-treated cells after IL-1β treatment.
(I) IP assay to detect interaction between PSD95 and ubiquitin.
(J) Ubiquitination of PSD95 in CBS-depleted cells after IL-1β treatment.
(K) Western blot analysis of PSD95 after treatment with a proteasomal inhibitor, MG132, following IL-1β treatment.
(L and M) Measurement of PSD95 level after treatment Siah RNAi by western blot (L) and confocal microscopy (M).
(N) Analysis of the number (a), density (b), and size (c) of PSD95 after hcy treatment.
(O) Ubiquitination assay of PSD95 in cells overexpressing either Siah or siah-delta RING domain after treatment with GYY4137.
(P) Ubiquitination assay of PSD95 in cells overexpressing either GAPDH or GAPDH-C150S following treatment with GYY4137. Please see Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 Depletion of CBS Stabilizes PSD95 Level
(A) Confocal microscopic analysis to detect intensity of WSP1 in the cortex of CBS+/− mice.
(B) Maleimide assay to detect sulfhydration of GAPDH both in cbs+/+ and cbs+/− mice after IL-1β (10 ng) injection.
(C and D) IP assay to detect interaction between Siah and GAPDH (C) and Siah and PSD95 (D) in both cbs+/+ and cbs+/− mice after IL-1β (10 ng) injection.
(E and F) Western blot hybridization (E) and confocal microscopic analysis (F) to detect PSD95 level in the IL-1β-injected cbs+/+ and cbs+/− mice.
(G) Analysis of spine number in cbs+/+ and cbs+/− mice after Golgi-Cox metal impregnation.
(H) A model represents how IL-1β leads to spine loss. Briefly, IL-1β induces intracellular H2S due to an increase in the level of protein level of CBS, which in turn essentially sulfhydrates GAPDH. Then, the modified GAPDH stabilizes Siah, which interacts with PSD95 and results in the degradation of PSD95 by ubiquitination. The loss of PSD95 leads to spine loss. ∗p < 0.01, n = 3, mean ± SEM, one-way ANOVA. Please see Figure S4.
Molecular Cell  , DOI: ( /j.molcel )
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