Flag tag f1804

Dulbecco’s modified Eagle’s medium (DMEM), minimum essential medium, EGF, LPA, verteporfin, crystal violet, poly-L-lysine, and anti-FLAG M2 Affinity agarose gel (A2220) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine 2000, Lipofectamine RNAiMAX, Opti-MEM I, DAPI, goat serum, and Alexa Fluor-conjugated secondary antibodies were from Thermo Fisher Scientific (Waltham, MA, USA). UNC3230, AR7, and 6AN were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies to α-tubulin (T5168), β-actin (A5316), vinculin (V4505), and FLAG-tag (F1804) were obtained from Sigma-Aldrich. Antibodies to HA-tag (#3724), Myc-tag (#2278), V5-tag (#13202), LATS1 (#9153), phospho-LATS1 Ser909 (#9157), YAP (#4912), phospho-YAP Ser127 (#4911), Merlin (#6995), and PIP5Kγ (#3296) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against HA-tag (sc-7392), lamin B1 (sc-374015), Hsc70 (sc-7298), GFP (sc-9996), and GAPDH (sc-47724) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

M. smegmatis expressing different plasmids was grown to an OD₆₀₀ of 0.8–1.2 in LB with or without Tween-80. Bacteria were pelleted, washed with PBS with 1% bovine serum albumin (BSA; Sigma), and incubated for 2  hours with antibodies recognizing the FLAG-tag (F-1804; Sigma-Aldrich), Strep-tag (NBP2-41073; Novusbio), or influenza virus hemagglutinin tag (2–2.2.14; Thermo Scientific). After washing with PBS with 1% BSA, bacteria were incubated with secondary goat anti-mouse IgG (A-21235; Thermo Fisher) or secondary goat anti-rabbit IgG (A-21244; Thermo Fisher) conjugated to Alexa 647 antibodies for 30 minutes. After washing with PBS with 1% BSA, bacteria were analyzed by flow cytometry (Attune NxT; Thermo Fisher). As a control, bacteria were incubated only with secondary antibodies. Where stated, the surface protein fraction of cells was extracted with 0.5% Genapol in PBS, after culture harvesting, afterwhich cells were washed once in PBS and labelling was done as stated above.