Rabbit anti phospho paxillin

The cells seeded in hydrogel microwells were fixed with 3% paraformaldehyde (Sigma‐Aldrich) for 20 min, permeabilized with 0.1% Triton X‐100 (Sigma‐Aldrich) for 15 min, and blocked with 2% BSA (Sigma‐Aldrich) or 5% Normal Horse Serum (Invitrogen) in PBS for 1 h to prevent nonspecific binding of the antibody used. After blocking, the samples were incubated for 1 h at 37 °C with an appropriate primary antibody and then incubated at room temperature with a secondary antibody and fluorescent probe for 1 h. All antibodies and fluorescent probes were diluted in 0.1% BSA in PBS. Primary antibodies included rabbit anti‐VE‐cadherin antibody (160840, Cayman Chemicals, 1:150) and rabbit anti‐Phospho‐Paxillin (69363, Cell Signaling, 1:100). The secondary antibody was goat anti‐rabbit antibody conjugated with Alexa Fluor 488 (A‐11034, Invitrogen, 1:150 for VE‐Cadherin and 1:100 for Paxillin). Actin was labeled with Alexa Fluor 488 Phalloidin (A12379, Invitrogen), or Rhodamine Phalloidin (R415, Invitrogen). Nuclei were labeled with 4',6‐diamidino‐2‐phenylindole, dihydrochloride (DAPI) (D1306, Invitrogen). The cells were labeled directly on the micropatterned hydrogels in the glass cloning rings to avoid disrupting the cell organization and were stored in PBS.

The antibodies used for immunoblot analysis in this study are as follows. All antibodies were used at a dilution of 1:1000 in TBS-T (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, 0.2% [v/v] Tween-20) with 3% (w/v) BSA unless otherwise indicated. Rabbit anti-pTyr (Cat#8954), mouse anti-HisTag (Cat#2366), rabbit anti-Paxillin (Cat#12065), rabbit anti-phospho-Paxillin (Y118; Cat#2541), rabbit anti-phospho-p120-Catenin (Y228; Cat#2911), and rabbit anti-phospho-p120-Catenin (Y904; Cat#2910) primary antibodies were all purchased from Cell Signalling Technology. Mouse anti-Afadin (Cat#610732) and mouse anti-p120-Catenin (Cat#610133) primary antibodies were purchased from BD Transduction Laboratories. Mouse anti-PTPRM (PTPRU cross-reactive; Cat#sc-56959)^{21 (link)} primary antibody was purchased from Santa Cruz. Rabbit anti-PTPRK primary antibody was generated in a previous study^{3 (link)}. Mouse anti-alpha-tubulin (Cat#T6199) and anti-FLAG (Cat#F7425) primary antibodies were purchased from Sigma Aldrich. HRP-conjugated anti-mouse (Cat#711-035-151) and anti-rabbit (Cat#711-035-152) secondary antibodies (1:5000 in TBS-T) were purchased from Jackson Immuno-Research.