Fig. 2
From: RAD21 is the core subunit of the cohesin complex involved in directing genome organization
RAD21-loader interaction facilitates vermicelli-like structure formation by promoting cohesin loading. A Co-immunoprecipitation and immunoblot assay against SMC3 detect cohesin formation using (whole cell extract)WCE obtained from HeLa cells with endogenous RAD21 or wild-type RAD21-OE. 3 independent experiments. B Proteins extracted sequentially at different salt (NaCl) concentrations were immunoblotted with antibodies specific for RAD21, SMC3, H3 or ACTB. 3 independent experiments. C The distribution of RAD21 and SMC3 detected in the Western blot shown in A was quantified and normalized to the amount of ACTB and H3. The sample volumes are 1: 0.5: 0.4 (0.1 M: 0.5 M: 1 M). D Co-immunoprecipitation and immunoblot assay against SMC3 detect cohesin loading using 0.1 M and 0.5 M salt extract obtained from HeLa cells with endogenous RAD21, wild-type or LIS-Ala-mutant RAD21-OE. 3 independent experiments. E Representative images of iFRAP assay of HeLa cells transfected with LIS-Ala-mutant RAD21 (scale bar is 5 μm). 3 independent experiments. F The fluorescence signals of unbleached regions were normalized to the first pre bleach image and plotted (mean ± S.D., n = 14 per condition). G Super-resolution images of cohesin subunits in the absence of RAD21 and wild-type/LIS-Ala SMC1A or SMC3 over-expression (on top of the normal wildtype copies). Scale bar, 5 μm. 3 independent experiments. Deconvolution was applied to improve the imaging resolution and signal to noise ratio