Fig. 3: Centromeric cenBAF localization depends on Flfl.
a Immunostainings with αBAF antibodies (red) are presented in cells expressing GFP::CENP-CR (left) and GFP::CENP-CΔFIMR (right) upon CENP-C depletion (dsRNACENP-C) and in control dsRNALacZ cells. GFP signals are direct fluorescence. DNA was stained with DAPI. Scale bars are 2.5 μm. b Quantitative analysis of the results shown in a. Mean grey values per centromere of αBAF fluorescence are presented for control dsRNALacZ and dsRNACENP-C cells expressing the indicated constructs. Values correspond to a representative experiment out of five independent experiments showing equivalent results (N > 48; Kruskal–Wallis test, p-value: ns > 0.05, **p < 0.01, ***p < 0.001). c co-IP experiments with αBAF antibodies in extracts from control dsRNALacZ and dsRNACENP-C cells expressing GFP::CENP-CR (left) and GFP::CENP-CΔFIMR (right) (lanes 4). Lanes 3 are mock IPs with preimmune serum. Lanes 1 and 2 are 2% and 5% of the input, respectively. IPs were analyzed by WB using αGFP and αBAF antibodies. The position of MW markers (in kDa) is indicated. d Quantitative analysis of the results shown in c. The ratio of αCENP-C and αBAF signals normalized with respect to the corresponding control dsRNALacZ cells is presented for dsRNACENP-C cells expressing the indicated constructs. Results are the average of two independent experiments (two-tailed t-test, ***p-value < 0.001). e Immunostaining with αFLAG antibodies (green) in metaphase chromosomes from cells transiently expressing FLAG::BAF3E. Immunostaining with αCENP-C antibodies (red) is also presented. DNA was stained with DAPI. Scale bar is 5 μm. f As in e but for cells transiently expressing FLAG::BAF3A. g co-IP experiments with αBAF antibodies in extracts from control dsRNALacZ cells, VRK1/NHK1-depleted dsRNANHK1 cells and pMT-NHK1 cells overexpressing VRK1/NHK1 (lanes 3). Lanes 2 are mock IPs with preimmune serum. Lanes 1 correspond to 3% of the input. IPs were analyzed by WB using αCENP-C and αBAF antibodies. The position of MW markers (in kDa) is indicated. h Quantitative analyses of the results shown in g. The ratio of αCENP-C and αBAF signals normalized respect to control dsRNALacZ cells is presented for pMT-NHK1 and dsRNANHK1 cells. Results are the average of three independent experiments (error bars are SD; two-tailed t-test, *p < 0.05, ****p < 0.0001).
