Polyclonal rabbit anti actin

Cells were lysed in freshly prepared lysis buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% nonidet 40; 5 mM DTT; 10 μg aprotinin; 10 μg leupeptin; 10 mM PMSF; 10% glycerol) on ice, and centrifuged at 12,000 rpm for 15 min. The supernatant was saved as whole-cell lysate, and protein concentrations were measured using the Pierce BCA Assay Kit (Thermo Scientific). For immunoblotting, 50–100 μg of protein was separated on a 5% stacking and 8% separating SDS-PAGE gel. Protein was transferred in Tris-glycine buffer with 20% methanol onto nitrocellulose membranes, and blocked with 5% non-fat milk in TBST, pH 7.5. After blocking, membranes were incubated overnight at 4 °C with primary antibodies (polyclonal rabbit anti-STIL, 1:200; Santa Cruz Biotechnology; polyclonal rabbit anti-ACTIN, 1:3,000, Sigma; polyclonal rabbit anti-cleaved Caspase-3, 1:3,000, Abcam) diluted in fresh blocking solution. The membranes were washed with TBST, and then incubated for 1 hour at room temperature with secondary antibodies (goat anti-rabbit IgG conjugated with HRP, 1:3000 for STIL, 1:30,000 for ACTIN) diluted in fresh blocking solution. The membranes were washed with TBST, and protein expression was detected using the Super-Signal West Pico Chemiluminescent Substrate (Thermo Scientific).

Protein samples from NRC and ARC cultures were prepared by scraping cells from the dish using a cell scraper and by subsequent lysis in a protein loading buffer at 90°C for 10 minutes. Protein samples were separated by SDS-PAGE using pre-cast NuPage 4–12% Bis Tris gels (Invitrogen) and blotted onto PVDF membrane (Milipore). After the transfer, membranes were blocked in TBST buffer supplemented with 5% milk (Sigma-Aldrich) for 1 hour at room temperature, followed by overnight incubation with primary antibody at 4°C. The following antibodies diluted in TBST + 5% milk were used: polyclonal rabbit anti-Ms1 (aABD2chn, 1:25000) polyclonal rabbit anti-actin (Sigma-Aldrich, 1:200), polyclonal rabbit anti-lamin A (Sigma-Aldrich, 1:200). After that, membranes were washed 3 times in TBST + 5% milk and HRP-conjugated anti-rabbit or anti-mouse Igs secondary antibody was added (1:5000 dilution, Santa Cruz). The secondary antibody was incubated for 1 hour at room temperature, followed by 3 washes in TBST buffer. To detect the antibody, chemiluminescent reagent (Invitrogen) was added and the blots were exposed to X-ray film (Fuji Film, RX NIF).

The antibodies used for immunostaining were: monoclonal mouse anti‐GALC clone mAb6 (described above), polyclonal rabbit anti‐cathepsin D (Calbiochem), polyclonal rabbit anti‐calreticulin (Pierce), polyclonal rabbit anti‐calnexin (Abcam), polyclonal rabbit anti‐REEP5 (proteintech), polyclonal goat AlexaFluor488‐conjugated anti‐mouse IgG (Life Technologies) and polyclonal goat AlexaFluor555‐conjugated anti‐rabbit IgG (Life Technologies). Antibodies used for immunoblotting were: monoclonal mouse anti‐FLAG (Sigma), monoclonal mouse anti‐GALC clones mAb1 and mAb2 (described above), polyclonal rabbit anti‐GALC (Abcam), polyclonal rabbit anti‐actin (Sigma), polyclonal goat IRdye680‐conjugated anti‐rabbit IgG (LiCor) and polyclonal goat IRdye800‐conjugated anti‐mouse IgG (LiCor).