Mouse anti gapdh antibody

After centrifugation at 15,000 g for 10 min, the unpurified supernatant and pellet (containing inclusion bodies) were obtained from the cells transformed with the H6-TAT-Cre or H6-E12-TAT-Cre plasmid. The pellets were washed with 20 mM Tris-HCl (pH 7.5), denatured completely with a 1% SDS solution by heating at 85°C, and sonicated until complete solubilization. The solution of a pellet in SDS was combined with the supernatant and designated as “total cellular protein.” The protein levels were measured using the BCA Protein Assay Reagent (Thermo Scientific). Thirty micrograms of proteins were separated by SDS-PAGE in a 12% gel and stained with Coomassie blue or transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 3% bovine serum albumin (BSA) for 1 h and probed with His Probe-HRP (1:5000 dilution in 3% bovine serum albumin, Thermo Scientific) for 1 h. A mouse anti-GAPDH antibody (Thermo Scientific) (1:5000 in Tris-buffered saline containing 0.1% of Tween 20 [TBST]) was incubated with the membrane for 1 h, followed by incubation with a horseradish peroxidase–conjugated mouse antibody (1:5000 in TBST, Thermo Scientific) for 1 h. The blots were developed by means of the Super Signal West Pico Chemiluminescent Substrate (Pierce).

U2OS cells transfected with pEGFP-N1-H2B or pEGFP-N1 and control cells were detached, washed in PBS, collected by centrifugation and lysed by re-suspension in 100 μl cell lysis buffer (Cell Signalling Technologies) supplemented with protease inhibitor cocktail (Roche) on ice for 15 min. Protein concentrations were determined by Bradford assay (Bradford 1976), using bovine serum albumin as standard concentrations. Proteins were separated by 15% SDS-PAGE and transferred onto Whatman Protran BA83 Nitrocellulose membrane using Towbin transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol pH 8). Proteins were detected using rabbit anti-H2B antibody (Cell Signalling Technologies), mouse/rabbit anti-H3 (abcam) and mouse anti-GAPDH antibody (Thermo Scientific), and corresponding HRP-coupled secondary antibodies (Cell Signalling Technologies). Protein signals were visualised using a myECL Imager (Thermo Scientific) and proteins bands were quantified using Image Studio Lite software (LI-COR).

Protein extraction was performed as previously described (Knop et al., 1999 (link)). Western blot analysis was performed using standard procedures (Popova, Wang, et al., 2021 (link)). Following primary antibodies were used: α‐syn mouse antibody (1:2000; BD Transduction Laboratory, USA), mouse anti phospho‐Ser129 α‐syn antibody (1:2000; Wako Chemicals, USA), mouse anti‐ubiquitin antibody (1:2000; Merch Millipore, USA), rat anti‐GFP antibody (1:1000; Chromotek, Germany), mouse anti‐GAPDH antibody (1:5000; ThermoFisher Scientific, USA), and mouse anti His6 antibody (1:1000; ThermoFisher Scientific, USA). Western blot quantifications of pixel density values were obtained from TIFF files generated from digitized x‐ray films (Kodak, USA), and analyzed with ImageJ software (NIH, Bethesda, USA). Sample density values are presented as ratios to the corresponding loading control and normalized to the control per blot. At least three independent experiments were performed for quantification of the signals.