Mouse igg kappa binding protein

Confluent monolayers of osteogenically differentiating MSC for 11 days were scraped in ice‐cold phosphate buffered saline (PBS), pelleted, and lysed in RIPA buffer (R0278; Sigma) with proteases and phosphatase inhibitors. Proteins in the supernatant were quantified with the Pierce^TM BCA assay (Thermo Fisher Scientific). For western blotting, equal amounts of proteins of each sample were boiled with 6X sample buffer (240 mM Tris pH 6.8, 40% glycerol, 8% SDS, 0.002% bromophenol blue, 0.002% β‐mercaptoethanol) for 5 min. The proteins were separated by 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked for 1 h with 5% nonfat milk, and incubated overnight at 4°C with primary antibodies (Table S2). Then, the membranes were incubated with peroxidase‐conjugated donkey anti‐rabbit immunoglobulin G (IgG) (dilution 1:25,000; Thermo Fisher Scientific) or mouse IgG kappa binding protein (dilution 1:1000, Santa Cruz Biotechnology). The signals were enhanced with chemiluminescence reagents (Amersham ECL Prime; GE Healthcare), and quantified with a Fusion camera and its Capt Fx Software (Vilber‐Lourmat).

Protein extracts from E15.5 female cortices were harvested by sonication in 1× RIPA lysis buffer and quantified for concentration using the Pierce BCA protein assay (Thermo Fisher Scientific). 25 μg of proteins were separated by SDS-PAGE and transferred onto PVDF membranes (0.2-μm-large pores) using the Bio-Rad system. Blots were blocked for one hour with 5% non-fat dry milk in TBS with 0.1% Tween-20 (TBS-T) and incubated overnight at 4°C in 3% BSA in TBS-T with antibodies against: BCL-2 (1:1,000; Cell Signaling, Danvers, MA, USA; #2772), BAX (1:1,000; Cell Signaling; #2772) or β-tubulin (1:2,000; Cell Signaling; # 2146). HRP-conjugated secondary antibodies, mouse IgG kappa binding protein (1:2,000; Santa Cruz; #sc-51602) or anti-rabbit IgG (1:2,000; Cell Signaling; #7074) were incubated for one hour, and chemiluminescence was detected using Clarity Western ECL Substrate on a Bio-Rad ChemiDoc MP imaging system. Using ImageJ software (http://rsb.info.nih.gov/ij/), all targets were analyzed densitometrically relative to β-tubulin loading control, and then were normalized to the average density of all bands within each blot to account for inter-run variability.