Hrp linked sheep anti mouse igg

Cells were harvested by scraping with a cell lifter and collected into microcentrifuge tubes. Samples were processed as previously described (Beringer et al., 2016 (link)). Specifically, cell pellets were washed with cold PBS and resuspended in 2.5x packed cell volume of PBS and an equal volume of 2x SDS lysis buffer (50 mM Tris HCl pH 7.5, 2 mM EDTA, 2% SDS). Samples were boiled for 10 min and sonicated for 5 min (High power, 30 s ON – 30 s OFF cycles). Protein extracts were quantified with Pierce BCA Protein Assay Kit. Protein extracts (1 to 10 ug) were loaded on 4–15% precast protein gels (Mini-PROTEAN TGX, Bio-Rad, #4561084). Membranes were incubated with rabbit cell cycle WB cocktail (Cdk pTyr15, H3 pSer10, actin) (Abcam, #ab136810), rabbit anti-SMC1A (Abcam, #ab133643), rabbit anti-SMC3 (Abcam, #ab128919), and mouse anti-vinculin 1:1000 (Sigma, #V9131) and with secondary antibodies sheep anti-Mouse IgG HRP-linked 1:1000 (GE Healthcare, #NA931), Donkey anti-Rabbit IgG HRP-linked 1:2000 (GE Healthcare, #NA934). HRP-derived signal was detected with Pierce ECL Western Blotting Substrate kit (Thermo Fisher Scientific, #32106) on an Amersham Imager 600 (GE Healthcare Life Sciences, # 29083461). Quantification of western blots was performed by Image Studio Lite 5.2 software (LI-COR), protein levels were normalized to Vinculin.