Pals1

Cells from three-dimensional cultures were fixed with 2% paraformaldehyde/PBS for 10 min, permeabilized in 0.5% Triton X-100/10% Goat serum/10% fish gelatin/PBS for 1 h and incubated overnight in primary antibodies. The primary antibodies were used at the following dilutions: Par6B 1/200 (Santa Cruz #sc-67393), aPKCι 1/100 (BD Transduction #610175), Par3 1/200 (Millipore #07-330), CD13 1/100 (Abcam #ab108382), CD13 1/50 (Sigma #HPA004625), E-cadherin 1/200 (Cell Signaling #3195 S), ZO-1 1/100 (Cell Signaling #8193), Ezrin 1/200 (Cell Signaling #3145), Pals1 1/100 (proteintech group #17710-1-AP), β1-integrin 1/200 (Abcam #ab30394), V5 1/200 (Thermo Fisher Scientific # R960-25), laminin 1/200 (Abcam #ab11575), α-Tubulin 1/200 (Sigma #T9026), Phalloidin 1/100 (Invitrogen #A34055), and endoplasmic reticulum (ER) marker-Calreticulin 1/200 (Abcam #ab92516). The secondary antibodies conjugated to Alexa488-Donkey anti-Rabbit IgG (Jackson ImmunoResearch Laboratories #711-545-152), Goat Anti-Mouse IgG (Cy3) (Jackson ImmunoResearch Laboratories #115-165-166) and Alexa647-Donkey Anti-Mouse IgG (Jackson ImmunoResearch Laboratories #715-605-151) were used at 1:750. DNA was detected with Hoechst dye 33258. Confocal imaging was performed using LSM700 from Zeiss with 20×/0.8NA or 40×/1.4NA objective lenses and processed using FIJI/ImageJ software.

Cells were lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 8, 0.15 M NaCl, 0.1% SDS, 1% NP-40, 1% sodium deoxycholate, 50 mM NaF, 5 mM orthovanadate, 1 mM DTT) supplemented with proteinase inhibitor cocktail (Sigma # 11836170001). Total proteins were separated by SDS-PAGE and transferred to Nitrocellulose membrane (Bio-rad # 1620115). The primary antibodies used were: aPKCι 1/1000 (BD Transduction #610175), α-Tubulin 1/5000 (Sigma #T9026), Par3 1/1000 (Millipore #07-330); Par6B 1/1000 (Santa Cruz #sc-67393), V5 1/5000 (Thermo Fisher Scientific #R960-25); flag 1/1000 (Delta Biolabs # DB125), CD13 1/1000 (Abcam #ab108382), CD13 1/500 (Sigma #HPA004625), Pals1 1/1000 (Proteintech Group #17710-1-AP), myc 1/1000 (Origene #A150121), GFP 1/1000 (Abcam #ab13970), and Phosphotyrosine clone 4G10 1/1000 (Millipore #05-321). For immunoprecipitation, cells were washed twice with ice-cold PBS and then lysed in NP40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl, 10 mM NaF, 1 mM NaVO₄, pH 8.0) containing a protease inhibitor cocktail and calyculin A. Lysates were precleared with MagnaBeads (Thermo Fisher Scientific #12321D) and then incubated with 2 μg of antibody or isotype control overnight at 4 °C. Antibodies were captured with MagnaBeads and washed three times with NP40 buffer.