Anti pias1 d33a7

Tissue samples were frozen in OCT and cryosectioned at 7 μM thickness (Leica). Each slide was fixed with 10% formalin for 15 min and washed twice with a 1% phosphate-buffered saline (PBS) solution. A solution of 5% goat serum in 1% PBS was applied to each slide for 45 min. A 1:50 ratio of mouse anti-COL7 primary antibody was added to a solution of 1% goat serum in 1% PBS, and split into primary antibody variations, maintaining a 1:200 ratio of anti-rabbit primary antibodies. Each primary antibody mixture was applied to the slides and incubated at 4°C overnight. Primary antibodies used include anti-PBRM1 (Bethyl, A700-019), anti-PIAS1 (D33A7) (Cell Signaling, 3550), and anti-COL7A1 (4D2) (Santa Cruz Biotechnology, sc-33710). The next day, the slides were washed three times with a 1% PBS solution. A 1:400 ratio of Goat anti-Mouse IgG (H + L) Alexa Fluor 594 (Thermo Fisher, A-11005) and a 1:400 ratio of Goat anti-Rabbit IgG (H + L) Alexa Fluor 488 (Thermo Fisher, A-11034) in a 1% goat serum in 1% PBS solution was applied to each slide and incubated at room temperature for 1 h in the dark. After, the slides were washed with 1% PBS, treated with Hoechst 33342 (10 μg/mL, Thermo) for 5 min, and then washed two more times with 1% PBS. Each slide was then mounted and imaged using the EVOS FL Auto 2 imaging system.

For immunoblot analysis, 10–30 μg of cell lysate was loaded per lane for SDS-page and then transferred to PVDF membranes. Blots were blocked with Odyssey Blocker PBS (Li-COR) + 2.5% BSA at room temperature for 1 h. Blots were then incubated with primary antibody at 4°C overnight and then with secondary antibodies (IRDye 680LT goat anti-mouse, LI-COR, 926–68020, 1:15,000, and/or IRDye 800CW goat anti-rabbit, LI-COR, 926–32211, 1:15,000) at room temperature for 1 h. The blots were imaged using Li-COR Odyssey CLx (LI-COR). Primary antibodies used for western blotting include anti-PBRM1 (Bethyl, A700-019), anti-PIAS1 (D33A7) (Cell Signaling, 3550), anti-ARID2 (D8D8U) (Cell Signaling, 82342), anti-BRG1 (G-7) (Santa Cruz Biotechnology, sc-17796), anti-ARID1A (PSG3) (Santa Cruz Biotechnology, sc-32761), anti-BRD7 (Bethyl, A302-304A-T), anti-HA (C29F4) (Cell Signaling, 3724), and anti-Lamin A/C (E−1) (Santa Cruz Biotechnology, sc-376248).