Enhanced chemiluminescence

Protein lysates from HaCaT cells and skin tissues were collected in ice-cold lysis buffer (Bio-Rad). Protein concentrations in cell lysates were determined using a DC kit (Bio-Rad). These samples were separated by SDS-PAGE electrophoresis and transferred onto a PVDF membrane. Following protein transfer, nonspecific sites were blocked with 5% nonfat dry milk in Tris buffered saline plus Tween-20 for 1 h at room temperature. Membranes were then incubated with primary antibodies overnight at 4°C. The next day, blots were washed three times for 10 min each on an orbital shaker, then membranes were incubated with HRP-conjugated secondary antibodies for 1.5 -2.0 h. Protein bands were visualized with an iBright1000 imaging system with the help of enhanced chemiluminescence according to the manufacturer’s instructions (Santa Cruz Biotechnology, Dallas, TX, USA).

Cells lines were washed with PBS and lysed at 0°C for 30 min using lysis buffer (120 mM NaCl, 30 mM KCl, 0.1% DTT, 0.5% Triton X-100) supplemented with protease and phosphatase inhibitor cocktail (Halt Protease Inhibitor Cocktail/ Halt Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific Inc.). Cell lysate was sonicated at 4°C for 10 sec and subsequently centrifuged at 15 000x g for 20 min. Supernatant was carefully removed and protein content was measured by the Bradford method (Bio-Rad, Hercules, CA); and the supernatants were stored at 80°C.
Equal amounts of proteins extracts were separated on a 4–15% SDS PAGE precast gel (Bio-Rad), and transferred to a nitrocellulose membrane followed by immunoblotting.
Rabbit monoclonal antibody against BRCA1 (clone D-20, Santa Cruz) was used at a final concentration of 1μg/ml. Goat monoclonal antibody against gelsolin (clone C-20, Santa Cruz) was used at a final concentration of 1μg/ml.
HRP-conjugated γ-Tubulin (clone C-20, Santa Cruz) was used at a final concentration of 1μg/ml to ensure equal amount of protein loading.
The signal was detected using anti-goat horseradish peroxidase-conjugate secondary antibodies, and developed by enhanced chemiluminescence (Santa Cruz)

Proteins from the cell lysates were isolated using radioimmunoprecipitation assay buffer (Sigma Aldrich, St. Louis, MO, USA) supplemented with a cocktail of protease inhibitor (Roche), and the protein concentration was determined using a BCA kit (Thermo Scientific, Carlsbad, CA, USA) according to the manufacturer’s protocol. Protein samples (20–35 ng) with loading buffer were loaded into the SDS-polyacrylamide gel, followed by their boiling for 5 min at 95 °C. After the electrophoresis and transferation of the proteins on 0.2 µm nitrocellulose membranes (GE Healthcare, Munich, Germany), they were then blocked for 1 h and incubated with primary antibodies overnight. After washing them three times with TBST, the membranes were incubated with secondary antibodies for 1 h and washed again. Enhanced chemiluminescence (Santa Cruz Biotechnology, Dallas, TX, USA) was used for signal detection. Information about the primary and secondary antibodies applied in this study is shown in Supplementary Table S2.

Total protein was quantified by the BCA protein assay. An equal amount of total protein (20 μg) was heat denatured, resolved by 12.5% SDS-polyacrylamide gel electrophoresis, and transferred to the PVDF membrane. After blocking with 5% nonfat milk, the blot was probed with primary antibodies for RPC (PAX6) and photoreceptor (RHO) markers (Supplementary Table 1) for 18 hours at 4°C, followed by the respective horseradish peroxidase- (HRP-) conjugated secondary antibodies. The signals were detected by enhanced chemiluminescence (Santa Cruz Biotechnology, Inc.) and imaged by the ChemiDoC XRS⁺ system (Bio-Rad). GAPDH was used as a housekeeping protein for normalization.

For immunoprecipitations, 6x10¹⁰ of MCF-7 and HCC1937 Cells were harvested and lysed in lysis buffer (20 mM NaCl, 30 mM KCl, 0.1% DTT, 0.5% Triton X-100, supplemented with protease and phosphatase inhibitor cocktail, Halt Protease Inhibitor Cocktail/ Halt Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific Inc.); cell lysates were clarified by centrifugation at 18,000xg for 30 min. BRCA1 was immunoprecipitated with 10 ng of anti-BRCA1 monoclonal antibody (clone D-20, Santa Cruz) during an overnight incubation with 1 ml of total cells extract (0.5 μg/μl). 20 μl of resuspended volume of Protein A/G PLUS-Agarose (Santa Cruz) were added to the mixture and incubated at 4° C on rotating device for 2 hour. Immune complexes were collected by low speed centrifugation, washed three times in 1 ml lysis buffer, and boiled in 20 μl of SDS loading buffer; denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (4–15% Bio-Rad precast gel). Proteins were transferred to Nitrocellulose membrane, which was blocked in 5% nonfat milk, 150 mM NaCl, 10 mM Tris (pH 8.0), and 0.05% Tween. Immunoblots were performed with rabbit anti-ATF1 antisera at 1 mg/ml (C-20 Santa Cruz.) and developed by enhanced chemiluminescence (Santa Cruz).