O8264

The interaction between PLXND1 and ORAI1 was assessed as previously described (Lin and Lai, 2017 (link)). The EEC lysates in the immunoprecipitated (IP) lysis buffer (pH 7.4, 0.025 M Tris, 0.15 M NaCl, 0.001 M EDTA, 5% glycerol) were incubated with antibodies or IgG overnight at 4°C in the dark. The antibodies used for IP included anti-PLXND1 (ab28762, Abcam) and anti-ORIA1 (O8264, Sigma) to immunoprecipitate PLXND1 and ORAI1, respectively. The immunoprecipitated protein complexes were collected by centrifugation at 10,000 × g and washed with an immunoprecipitate elute (26,149, Thermo Scientific Pierce Co-IP kit) to remove unbound immune complexes. The bound immune complex was then analyzed by SDS-PAGE using anti-PLXND1 and anti-ORIA1, respectively.

Cells were lysed in PBS containing 2% NP-40 and lysates were subjected to SDS-PAGE (BioRad Miniprotein) and proteins were blotted onto polyvinylidene difluoride membrane (PVDF, Amersham Pharmacia Biotech). The primary antibodies anti-NF-κB p65 (#sc-8008, dilution 1/500) from Santa Cruz Biotech, phospho-NF-κBp65 (#3033, dilution 1/500) from Cell signaling, TRPC1: rabbit anti-TRPC1 (1:500, T8276; Sigma-Aldrich, United States), STIM1: rabbit anti-STIM1 (1:500, 4916S; Cell Signaling, United States) and Orai1: rabbit anti-Orai1(1:250, O8264; Sigma-Aldrich, United States) were revealed by HPR-conjugated secondary antibodies (Amersham Pharmacia Biotech) and enhanced chemiluminescence (Pierce ECL Plus, Thermo Scientific) according to the manufacturer’s instructions. Images were acquired with a Genegnome system and GeneSyssoftware (Syngene) (17 (link)–19 (link)).

Cells were dissected and homogenized in lysis buffer (RIPA: protease inhibitor cocktail = 100:1). Whole cell lysates were analyzed in a western blot experiment according to standard protocols. Antibodies for immunoblotting were as follows: Orai1 rabbit polyclonal antibody (13130-1-AP, Proteintech, 1:100 dilution), rabbit anti-ORAI1 (O8264, Sigma, 1:1000 dilution), TRPC1 rabbit polyclonal antibody (19482-1-AP, Proteintech, 1:500 dilution), GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech, 1:10,000 dilution), Peroxidase AffiniPure goat anti-mouse IgG (H + L) (115-035-003, Jackson ImmunoResearch, 1:10,000 dilution), Peroxidase AffiniPure goat anti-rabbit IgG (H + L) (111-035-003, 1:10,000 dilution, Jackson ImmunoResearch). Proteins were visualized using enhanced chemiluminescence. The images were acquired by Tanon 4200SF (Tanon). Immunofluorescence microscopy of eIF4E-p was performed 24 h after femtoSOC. Cells were fixed with 5% paraformaldehyde and permeabilized with 0.5% Triton X-100 (1848549B, Life Technologies). The cells were then incubated with an anti-eIF4E antibody (phosphor S209, 1 μg/mL, ab76256, Abcam) overnight at 4 °C. The sample was washed with PBS for 5 min and incubated with diluted secondary antibody anti-Rabbit IgG (H + L) (Alexa Fluor 488, 2 μg/mL, ab150077, Abcam) for 1.5 h at room temperature.