Alexa 488 a11008

Cells were seeded onto 4-chamber slide wells at a concentration of 20,000 cells per well. Clustered cells were attached using a cytospin. Cells were fixed with 4% paraformaldehyde and permeated with 1% Triton X-100 in PBS for 15 min at room temperature. Cells were then washed three times for 5 min each with 0.05% PBS and blocked with 10% bovine serum albumin (BSA) in PBS for 60 min. Cells were then washed again and primary antibody was added overnight at 4°C (CD81 Millipore HPA007234 [RRID:AB_1846333] 4 µg/ml and CD44 MA513890 [RRID:AB_10986810] 2 µg/ml). After washing, secondary antibody was added for 60 min at room temperature (Texas Red T862 Thermo Fisher (RRID:AB_2556781) and Alexa-488 A11008 Thermo Fisher (RRID:AB_1431650)). Finally, the cells were washed, and a cover slide was placed with mounting media. The slides were imaged using Nikon A1R (A) Spectral.

For SGK1.1 detection we used a rabbit polyclonal antibody produced in our laboratory (Martin-Batista et al., 2021 (link)). SGK1 was detected using rabbit anti-SGK1 polyclonal antibody (Abcam, ab43606). Kv7.2 was detected with rabbit anti-Kv7.2 polyclonal antibody (Abcam, ab22897). YFP-tagged SGK1.1 and GFP-tagged Kv7 subunits were detected using mouse anti-GFP monoclonal antibody (Abcam, ab290). Nedd4-2 was detected with a rabbit polyclonal antibody from Cell Signaling (4013S). GAPDH was detected with a mouse monoclonal antibody (Abcam, 9484). Secondary antibodies conjugated to fluorophores were obtained from Thermo Fisher Scientific (Alexa 594, A-11042; Alexa-488, A-11008).