Anti aid

Protein expression was analyzed 72 hr post-stimulation for switching cells unless otherwise indicated. Briefly, 0.5 million cells were suspended with RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH8.0, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Protease Inhibitors) and incubated at 4℃ for 30 min, and following centrifuge to remove the thick DNA, the whole-cell lysis was boiled in protein loading buffer for SDS-PAGE. Immunoblotting was performed with the appropriate primary and secondary antibodies. For FLAG-RNaseH2B immunoprecipitation, total cell lysates were prepared in RIPA buffer and incubated with a desired antibody and appropriate protein A/G-agarose beads at 4°C overnight with gentle agitation. Beads were washed three times with lysis buffer, and immunocomplexes were eluted by boiling in SDS sample buffer for 5 min before loading. Anti-AID (Thermo Fisher ZA001) at 1:500 dilution, anti-β-actin (ABclonal, AC026) at 1:100,000 dilution, anti-FLAG (ABclonal, AE005), goat anti-mouse Alexa Fluor 680 secondary antibody (A21057), and goat anti-rabbit Alexa Fluor 790 secondary antibodies (A11367) were employed.

Samples were collected and placed on ice in a lysis solution [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol] containing 0.5% SDS and 2 mM PMSF with a protease inhibitor cocktail (Sigma P-8340, 1:100). Cellular proteins were resolved on a 12.5% SDS–PAGE gel. The membrane was incubated for 1 h at room temperature in 5% skim milk in PBS with 0.05% Tween-20 (PBST), and the membrane was probed with anti-PCNA PC10 (Ref # sc56; Santa Cruz), anti-alpha tubulin (Ref# MA1-80017; Thermo Fisher Scientific), anti-actin (Ref #MA1-744; Thermo Fisher Scientific), anti-Vinculin (clone 7F9, Ref# 14-9777-80; eBioscience), anti-AID (Ref #14-959-82; Thermo Fisher Scientific), anti-polη (Ref# ab17725; Abcam), anti-FancD2 (Ref# sc20022; Santa Cruz), anti-USP1 (Ref # ab108104Ref; Abcam), anti-Msh2 (Ref #A300-451A; Bethyl), and anti-Msh6 (Ref # A300-022A; Bethyl) antibodies. Immunoreactivity was detected using a horseradish peroxidase-conjugated secondary antibody.