Sc 136239

Western blots were performed as described previously^{19 (link),20 (link),47 (link)} using NuPage 4–12% gels and the following antibodies. Uncropped, original blots are provided in Supplementary Fig. 1.
The following primary antibodies were used: anti-TRPV4 antibody (raised in mouse; 1B2.6; Millipore Sigma; MABS466; 1:1,000), anti-ARP3 (mouse; FMS338; Abcam; ab49671; 1:5,000), anti-ARPC4 (rabbit; Abcam; ab217065; 1:2,000), anti-integrin β1 antibody (rabbit; Cell Signaling; 4706S; 1:1,000) and anti-NHE1 (raised in mouse; 54; Santa Cruz Biotechnology; sc-136239; 1:200). GAPDH was used as a loading control (rabbit; 14C10; Cell Signaling; 2118S; 1:5,000).
The following secondary antibodies were used: anti-mouse IgG, HRP-linked antibody (Cell Signaling; 7076S; 1:1,000) and anti-rabbit IgG, HRP-linked antibody (Cell Signaling, 7074S; 1:1,000).

AP-1 cells (untransfected or stably expressing wt- or 4G-NHE1 as indicated) were grown on 12 mm round glass coverslips, fixed in 2% paraformaldehyde (15 min room temperature, 30 min on ice), washed in TBST, permeabilized for 5 min (0.5% Triton X-100 in TBS), blocked for 30 min (5% BSA in TBST), incubated with primary antibody against NHE1 (Santa Cruz Biotechnology sc-136239 (54); 1:100 dilution in TBST + 1% BSA) o/n at 4 °C. The day after, coverslips were washed 3 times in TBST + 1% BSA and incubated with the appropriate conjugated secondary antibody (1:600 in TBS + 1% BSA) in combination with rhodamine-conjugated phallodin (ThermoFisher, #R415) to label F-actin (1:100 in TBS + 1% BSA) for 1 h, followed by washing in TBST + 1 % BSA, and mounting in N-propyl-gallate mounting medium (2% (w/v) in PBS/glycerol). To stain nuclei, DAPI was added for 3 min following incubation with the secondary antibody. Cells were visualized using the 60X/1.35 NA objective of an Olympus Bx63 epifluorescence microscope. No or negligible labeling was seen in the absence of primary antibody or in untransfected AP-1 cells. Overlays and brightness/contrast adjustment was carried out using Adobe Photoshop software. No other image adjustment was performed.