Catalog no 7074s

The cellular proteins were prepared from cells growing on a 6-well plate after treatments of BNAG1, or BNAG2 or NAG using a commercial RIPA buffer (5X) (Catalog No. AAJ62524AD, Thermo Fisher Scientific) containing a protease inhibitor cocktail (Catalog No. sc-29130, Santa Cruz Biotechnology Inc., Dallas, TX, USA) and 1 mM phenylmethylsulfonyl fluoride (Catalog No. sc-482875, Santa Cruz Biotechnology Inc.). Then the protein concentration was determined using the Bradford protein assay kit (Catalog No. 5000201, Bio-Rad, Hercules, CA, USA). SDS-polyacrylamide gel electrophoresis, protein transfer to nitrocellulose membranes (Catalog No. 88025, Thermo Fisher Scientific), incubation of membranes with primary antibodies against mouse iNOS and Cox-2 (Catalog No. NB300-605 and NB110-1948, both from Novus Biologicals, LLC), and horseradish peroxidase (HRP)-conjugated secondary antibody (Catalog No. 7074S, Cell Signaling), and visualization of protein bands were performed according to the procedures described previously [18 (link)]. β-actin (Catalog No. sc-47778, Santa Cruz Biotechnology Inc.) was used as the loading control. All Western blot assays were conducted in duplicates.

Lenses at stage P15 from Tdrd7−/− and control mice were subjected to RIPA lysis buffer to prepare lens lysate. Protein lysate concentration was determined by BCA assay following the manufacturer’s instructions (Pierce BCA Protein Assay Kit, Thermo Fisher Scientific). Equal concentrations of the protein lysate for Tdrd7−/− and control lenses were loaded on a 7% polyacrylamide gel and then transferred onto PVDF membrane. The membrane was blocked with 5% milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at room temperature, followed by overnight incubation at 4°C with primary antibody for HSPB1 (Abcam, Catalog No. ab2790, used at 1:200 diln.) and for beta-actin (loading control) (Abcam, Catalog No. ab8227) in the blocking buffer. The membrane was then washed and probed with a secondary HRP-conjugated antibody (Cell Signaling, Catalog No. 7074S) for 1 h at room temperature, followed by washes and imaging.