Gam007

After treatments, the cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (Beyotime, Beijing, China) with protease inhibitors, sonicated for 15 seconds and centrifuged at 12,000 g for 15 minutes at 4 °C. The protein concentrations were determined by a BCA protein assay kit (Beyotime, Beijing, China), and then the protein samples were mixed with loading buffer and heated at 100 °C for 10 minutes. Equal amounts of protein samples were loaded per lane, separated by 10% SDS-PAGE, and transferred onto PVDF membranes (Millipore), which were then blocked with 5% non-fat milk for 3 hours. The PVDF membranes were then probed with different antibodies: rabbit anti-LAMP2 (1:1500, Abcam), rabbit anti-Beclin1 (1:1000, CST), rabbit anti-LC3 (1:1000, CST), rabbit anti-cleaved-caspase3 (1:1000, CST) and mouse anti-β-actin (1:5000, Sungene Biotech). After the primary antibody binding, the membranes were incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit (1:8000, MultiSciences, GAR007, China) or goat anti-mouse IgG (1:8000, MultiSciences, GAM007, China) at room temperature for 1 hour. The reactive proteins were detected by an ECL chemiluminescence system (GE Healthcare, Piscataway, NJ, USA), the band intensities were quantified with Quantity One software, and the results were normalized to β-actin.

Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany, 11873580001). Protein concentration was determined. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a poly(vinylidene fluoride) (PVDF) membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20 the membrane was incubated with specific primary antibodies, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies. Signals were detected using an enhanced chemiluminescence reagent (Millipore, Darmstadt, Germany, WBKLS0500) and subjected to chemiluminescence instrument (Beijing Sage Creation Science Co. Ltd). Poly ADP-ribose polymerase (PARP) antibody (Promega, Madison, WI, G734A, 1:1000 dilution), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Bioworld, Minneapolis, MN, MB001, 1:5000 dilution), HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000 dilution).

The cells were collected using a scraper, mixed evenly, dissolved on ice using lysate for 40 min, placed into a pre-cooled EP tube and centrifuged at 4°C 14000 rpm for 15 min. Protein concentration was detected using BCA kit (Life-iLab, Shanghai, China). Following electrophoresis at 100 V for 40 min and 80 V for 1 h, membrane transference was carried out at 250 mA and lasted for 2 h. The membrane blocking was performed using 5% skimmed milk at room temperature for 1 h, followed by addition of primary antibodies, vibrated for 1 h at room temperature. The membrane followed three cycles of washing using Tris-buffered saline with Tween 20 (TBST), 10 min each time. Secondary antibodies were subsequently supplemented for 1 h incubation at room temperature and rinsed twice using TBST [29 (link)]. Photographs were taken for grayscale analysis via Image J software. The antibodies were listed as following: BMP2 at 1:2000 rate (BM0231, IGEE), GAPDH at 1:1000 rate (5174S, CST), GATA6 at 1:1000 rate (BM16634, IGEE), MGP at 1:1000 rate (10,734-1-AP, PROTEINTECH), pp38 at 1:1000 rate (4511 T, CST), p38 at 1:1000 rate (8690S, CST), HPR Goat anti-rabbit IgG at 1:2000 rate (BMS041, IGEE) and goat anti-mouse IgG at 1:8000 rate (GAM007, MultiSciences).

Cells were lysed in RIPA buffer containing a protease inhibitor cocktail (Roche, Mannheim, Germany, 11873580001). Protein concentration was determined. Equal amounts of protein were separated by SDS-PAGE and electrophoretically transferred onto a PVDF membrane (Roche, 03010040001). After blocking with 5% nonfat milk in Tris-buffered saline containing 0.1% Tween-20, the membrane was incubated with specific primary antibodies, followed by incubation with appropriate horseradish peroxidase–conjugated secondary antibodies. Signals were developed using an enhanced chemiluminescence reagent (Millipore, Darmstadt, Germany, WBKLS0500) and captured on an Alpha Innotech Fluor Chem FC2 imaging system (Alpha Innotech, San Leanardo, CA). Antibodies used in this study were: rabbit anti-β-ACTIN (Biosynthesis Biotechnology, Beijing, China, bs0061R, 1:1000), rabbit anti-HMGB1 (Abcam, Hong Kong, China, ab191583, 1:1000), rabbit anti-AMPK/pAMPK (Cell Signaling Technology, Danvers, MA, #2532 / #2531, 1:1000), rabbit anti MAVS (Abcam, ab31334, 1:500) and HRP-conjugated secondary antibodies (Multisciences, Hangzhou, China, GAR007 and GAM007, 1:5000).

We established a protein IP assay to detect anti-EJ in patients with myositis. Human HEK293 cells (ATCC CRL-1573) were grown in DMEM supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Invitrogen) in a humidified atmosphere of 5% CO₂ at 37°C. Transient transfections were carried out using Megatran 2.0 (TT200003, Origene, USA) with pENTER-flag- GlyRS (glycyl tRNA synthetase) plasmid (CH810182, Vigene Biosciences, USA). After 48 hrs, cells were lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) for western blot or protein immunoprecipitation. IP was performed according to protocols with minor modifications [16 (link), 17 (link)]. Immunoprecipitated antigens were solubilized in 1× SDS-PAGE loading buffer and separated by 10% SDS-PAGE and transferred to PVDF membrane. The membrane was then detected with anti-flag antibodies (1:1000, F1804, Sigma). The secondary antibody used was HRP-conjugated anti-mouse IgG (1:10000, GAM007, Multi Science).

The rat hippocampus was collected immediately after the animals completed the behavioral tests. Proteins of the tissues were extracted and the concentrations determined by BCA Protein Assay Kit (CWBIO, Beijing, China). Each sample contained with 40 μg of protein was separated by 10 or 12% SDS-PAGE and then transferred to nitrocellulose membranes (Millipore, MA). The following primary antibodies were incubated with the PVDF membrane at 4°C overnight. PBR (1:7,000 dilution, ab92291; Abcam), ATG7 (1:7,000 dilution, ab133528; Abcam), ATG5 (1:7,000 dilution, ab108327; Abcam), LC3B (1:2,000 dilution, ab192890; Abcam), p62 (1:7,000 dilution, ab109012; Abcam) and β-actin (1:7,000 dilution, aa128; Beyotime Biotechnology). After that, rabbit anti-goat immunoglobulin G (IgG) (H + L) horseradish peroxidase (HRP) (1:5,000 dilution, ZB2306; ZSGB-BIO), goat anti-rabbit immunoglobulin G (IgG) (H+L) HRP (1:7,000 dilution, GAR007; MultiSciences) or goat anti-mouse IgG (H+L) HRP (1:7,000 dilution, GAM007; MultiSciences) were incubated with the membrane for 2 h at room temperature. Bands were visualized with enhanced chemiluminescence (ECL) detection reagents (CWBIO, Beijing, China) using an ECL reagent. The relative band intensity was measured by Image-Pro Plus software.