1. Figure e-1 A. Detection of anti-TIF1-γ antibody.
150kDa
100kDa
50kDa
input P HC P P P P P P7 P P P10 P P12
yes yes yes yes yes yes yes yes yes yes yes no
Lane
patient
anti-TIF1-γ Ab
Cancer
P=patient 　　HC=healthy control
B. ELISA of anti-HMGCR antibody
corrected OD value
Autoimmune
disorders
of the CNS
(n = 25)
Autoimmune
disorders
of the PNS
(n = 18)
Other
neurological
disorders
(n = 29)
Healthy
control
(n = 20)
IIMs
(n = 343)

2. A. Detection of anti-TIF1-γ antibody.
The biotinylated recombinant TIF1-γ protein was immunoprecipitated using sera from different patients (lanes 2, and 4 to 13) or healthy normal subjects (lane 3). The eluated precipitates were incubated with sample buffer and separated by SDS-PAGE. Then, the biotinylated TIF1-γ protein was visualized using the Western blue substrate (Promega) after transferring onto polyvinylidene difluoride (PVDF) membranes. Lane 1 (input): 0.5 μl of biotinylated recombinant TIF1-γ protein solution used for the immunoprecipitation without adding sera in sample buffer. Lanes 2 to 13: Five microliters of eluted precipitates in sample buffer. We visually determined the immunopositivity for the 155 kDa band in each lane. None of the sera from healthy control subjects precipitated the recombinant TIF1-γ protein at a visual level.
For the detection of the anti-TIF1γ antibody, the recombinant TIF1-γ protein was produced by an in vitro translation method. TIF1-γ cDNA was obtained from Kazusa DNA Research Institute (Japan). We first amplified TIF1-γ cDNA using the following primers: TIF1-γ UP1,
5’-CCCCTCGAGGCCGCCATGGCGGAAAACAAAGGCGGCGGCGAG -3’ ;
and TIF1-γ/pBluescriptSK(+)DW1,
5’-GGGGCCCGGTACCCAATTCGCCCTATAGTGAG-3.
After gel purification, the PCR products of TIF1-γ were digested with XhoI and NotI and then inserted into the pTNT vector (Promega, Madison, WI), then digested with the same restriction enzyme. All constructs were verified by DNA sequencing. The biotinylated recombinant TIF1-γ protein (TnT products of TIF1-γ) was produced from the prepared vector containing full-length TIF1-γ by in vitro translation using a TNT T7 Quick Coupled Transcription/Translation System (Promega), in accordance with the manufacturer’s protocols.
For detecting the anti-TIF1-γ antibody in human sera, immunoprecipitation was performed using the TnT products of TIF1-γ by a previously described method 16 with minor modification. Briefly, 10 μl of patients’ sera and 190 μl of antibody (Ab)-binding and washing buffer (0.1 M Na-phosphate buffer with 0.02% Tween 20, pH 7.4) were mixed and incubated with 50 μl of magnetic beads (Dynabeads proteinG; Life Technologies) for 30 min. After washing the Dynabeads proteinG once with the Ab-binding and washing buffer, 10 μl of biotinylated TnT products and 190 μl of immunoprecipitation buffer (phosphate-buffered saline containing 0.5% Nonidet P-40, pH 7.2) were mixed and incubated with the beads for 30 min. The beads were washed three times using washing buffer (0.1 M Na-phosphate buffer, pH 7.4) and eluted with 18 μl of elution buffer (50 mM glycine buffer, pH 2.8). The eluted solution was incubated with 4.5 μl of sodium dodecyl sulfate (SDS) sample buffer (0.25 M Tris‐HCl, pH 6.8, 16% SDS, 40% glycerol, 0.20 M DTT, and 0.4% bromophenol blue) at 70 ºC for 10 min, and 10 μl of the sample mixture was applied to 7.5% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). Then, proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The biotinylated TIF1-γ protein was subsequently detected using the Western blue substrate (Promega).

3. B. ELISA of anti-HMGCR antibody
ELISA of anti-HMGCR Ab was performed on the basis of the original method with some modification (Mammen et al. 2011). Ninety-six-well polystyrene ELISA plates (Corning Life Sciences, Lowell, MA, USA) were coated with 100 ng of a C-terminal recombinant HMGCR protein (Sigma, St. Louis, MO, USA) or phosphate buffer saline (PBS) as a control. After blocking with 1% bovine serum albumin (BSA) in PBS, patient serum diluted at 1:400 with 1% BSA in PBS was added as the primary antibody and allowed to stand for 120 min. After washing with PBS-T (0.05% Tween 20 in PBS), horseradish peroxidase-conjugated anti-human IgG-Fc antibody (MP Biomedicals, Solon, OH, USA) diluted at 1:500 with 1% BSA in PBS was added as the secondary antibody and allowed to stand for 90 min. The wells were washed again and the chromogenic reaction was developed with O-phenylenediamine in phosphate-citrate buffer. Optical density (OD) was measured at 490 nm (Microplate Reader Model 680, Bio-Rad Laboratories Inc., Hercules, CA, USA). The corrected OD was calculated in duplicate by subtracting the OD values of PBS as the control OD. The cut-off OD value was set as the mean +5SD of healthy control sera (broken line, 0.130).
ELISA of anti-HMGCR Ab was performed in 343 patients with available serum (six patients were not included because of their serum was not available), and 72 disease controls consisting of 25 patients with autoimmune disorders of the central nervous system, 18 patients with autoimmune disorders of the peripheral nervous system, and 29 patients with other neurological disorders. Anti-HMGCR Ab was positive in 18 patients including 8 CAM patients and 5 non-CAM3y patients (table e-2). One dermatomyositis patient in the non-CAM3y subgroup, who showed markedly increased high serum CK level (11000 IU/L), showed lowest positive OD (0.167).
None of the disease control patients were positive for anti-HMGCR Ab.