Mouse anti cortactin

Cells were fixed with 4% formaldehyde or ice-cold methanol and blocked with PBS/2% normal goat serum. Fixed cells were incubated with the primary antibodies rat anti-HA (Roche, 3F10; 1:300), rabbit anti-HA (Sigma-Aldrich, H6908, 1:300), mouse anti-Cortactin (MerckMillipore, 4F11; 1:500), mouse anti-E-cadherin (BD Biosciences, 610182, 1:1000) and rabbit anti-TKS5 (Santa Cruz, sc-30122, 1:100) as indicated in the text at 4°C overnight, followed by Alexa Fluor_ 488-conjugated donkey anti-rat IgG (1:500, A21208; Life Technologies), Alexa Fluor_ 350-conjugated goat anti-mouse IgG (1:500, A11045; Life Technologies), Alexa Fluor_ 594-conjugated goat anti-mouse IgG (1:500, A21135; Life Technologies) or Alexa Fluor_ 488-conjugated goat anti-rabbit IgG (1:500, A-11029; Life Technologies) for 1 h at room temperature and counterstained with DAPI (Molecular Probes).
F-actin was stained by Alexa fluor 488 phalloidin or Rhodamine phalloidin (1:100, A12379, R415, Life Technologies).

Gelatin coverslips were prepared as published previously [33 (link)]. Briefly coverslips were with treated with 50 μg/ml poly-L-lysine, then 0.5% glutaraldehyde, then 0.2% Oregon green gelatin (Molecular Probes, Eugene, OR, USA), then 5 mg/ml NaBH₄, with PBS washes done between each treatment. Rheumatoid synovial fibroblasts (7.5 × 10³) were plated on the coverslips in media with 10% FBS; incubated for 2, 5 or 24 hours; fixed with 3% formaldehyde; quenched with 0.15 M glycine; permeabilized with 0.2% Triton-X 100; blocked with 5% goat serum; and incubated with mouse anti-cortactin (1:200, clone 4F11; EMD Millipore, Billerica, MA, USA), mouse anti-vinculin (1:500, clone V284; EMD Millipore) and rabbit anti-paxillin pY118 (1:200, polyclonal; BioSource International, Camarillo, CA, USA), followed by washing and incubating with either goat anti-mouse rhodamine red or donkey anti-rabbit tetramethylrhodamine (1:250). Coverslips were mounted and viewed at 400× magnification with an inverted fluorescence microscope (Nikon Eclipse TE300; Nikon Instruments, Melville, NY, USA). Images were digitally acquired and processed with MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) and ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Cells were plated on 0.1% gelatin-coated glass coverslips. Following incubation, cells were washed with PHEM buffer (2 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, 60 mM PIPES, pH 6.9) and fixed for 20 min with cold PFA 4%. Next, cells were permeabilized with 0.1% Triton X-100 for 10 min, blocked with 1% bovine serum albumin in PHEM buffer for 30 min, and then incubated with mouse anti-cortactin (1:200, #05–180, RRID:AB_309647, Merck) and rabbit anti-Ser5-P-L-plastin (1:50) at 4 ºC overnight, followed by incubation with Alexa Fluor 405-conjugated goat anti-mouse IgG (1:250, #A31553, RRID:AB_221604, Thermo Fisher Scientific), Alexa Fluor 488-conjugated GFP booster (1:200, #gb2AF488-10, RRID:AB_2827573, Chromotek), Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:250, #A11037, RRID:AB_2534095, Thermo Fisher Scientific) and Alexa Fluor 633-conjugated phalloidin (1:50, #A22284, Thermo Fisher Scientific) or Alexa Fluor 568-conjugated phalloidin (1:50, #12380, Thermo Fisher Scientific) at room temperature for 1 h. Coverslips were mounted using Vectashield Anti-fade Mounting Medium (#H-1000, RRID:AB_2336789, Vector Laboratories, San Francisco, CA, USA) and image acquisition was performed using the Andor Spinning Disk Revolution system (CSU-W1; Andor Technology, Belfast, United Kingdom) based on a Nikon Ti microscope (Nikon, Tokyo, Japan) with an Andor iXon Ultra EMCCD camera.