Fig. 6

Flag-tagged Sgcg directly or indirectly associates with HA-CFP-tagged NKCC1 proteins. A Immunoblot of the most successful co-IP, showing more Sgcg-Flag sedimenting with the NKCC1 and NKCC1-cyto (bound), compared to the HA-CFP negative control; corresponding decreases were observed in supernatants after sedimentation (Unbound). Sgcg-Flag amounts in cell lysates also are shown (Input). Flag-tagged Sgcg, Sgcb, and Sgcd proteins were co-expressed with either HA-CFP-NKCC1 (lane 1), HA-CFP (lane 2), or HA-CFP-NKCC1 cytoplasmic domains (NKCC1-cyto, lane 3) in RH-30 cells and immunoprecipitated with anti-HA. Bound NKCC1 proteins were visualized with anti-HA (asterisks); bead-bound rabbit anti-HA heavy chain (IgG). B Densitometric quantification of Unbound/Input ratios of Sgcg-Flag from supernatant-depletion assays, such as those shown in C, and analysis by one-way ANOVA with Tukey’s multiple comparisons test. P values for the mean differences between HA-CFP control and HA-CFP-NKCC1 and HA-CFP-NKCC1-cyto were 0.12 and 0.02, respectively (*P < 0.05). Central bars, means; error bars, S.D. C Representative immunoblot for supernatant-depletion assays in B, showing decreases in band intensity in Unbound lanes (U), compared to Inputs (I). IP used rabbit monoclonal anti-HA (HA); Sgcg-Flag was visualized with rabbit anti-Flag (Flag); HA-CFP-tagged NKCC1 (lane 1), HA-CFP alone (lane 2), or HA-CFP-NKCC1 cytoplasmic domains (lane 3). D, E Representative images and quantification of PLA for the SC interaction with HA-CFP-NKCC1, HA-CFP-NKCC1-cyto, or the HA-CFP control in RH30 cells co-transfected with Flag-tagged Sgcg, Sgcb, and Sgcd. D In cells fixed without detergent pre-treatment to remove free cytosolic proteins (Figs. 4D and 5D), PLA speckles indicating proximity of anti-Sgcg and anti-HA were observed with all three HA-CFP constructs (Da–c). A brief detergent pre-extraction revealed persisting proximity of the SC complex with NKCC1 and NKCC1-cyto, but not with the HA-CFP tag alone (Dd–f, E). PLA speckles are pseudocolored yellow; DAPI-labeled nuclei in blue. Size bars, 20 μm. E Graph of PLA speckles per nucleus for pre-extracted cells. Cells with no PLA speckles were assumed to be untransfected and were not included. Horizontal bars, means from results combined from 2 experiments with the following total numbers of counted cells: NKCC1 (70), NKCC1-cyto (56), HA-CFP (65). Transfection efficiencies varied from 36% to 65%, with higher values for HA-CFP, despite a lower percentage of PLA speckles. ****P < 0.0001; P for NKCC1 versus NKCC1-cyto was 0.6140 (ns), from a one-way ANOVA with a Kruskal-Wallis multiple comparisons test