Image reader las 3000 lcd camera

For immunoprecipitation, 6 x His-tagged ORAI1 and 6xHis-tagged STIM1 or Vav1-GFP together with empty vector or FLAG-tagged WT CRACR2A-a, FLAG-tagged CRACR2A^E278D or CRACR2A^{R144G, E300*} was transfected into HEK293T cells. Transfected cells (2 × 10⁷) were lysed in lysis buffer (20 mM Tris-Cl, 2 mM EDTA, 135 mM NaCl, 10% (vol/vol) glycerol, 0.5% Igepal CA-630, protease inhibitor mixture, pH 7.5) and centrifuged at 100,000 x g for 1 hr before preclearing with protein G-Sepharose. Lysates were immunoprecipitated with anti-FLAG antibody-conjugated resin for 6 hr. Immunoprecipitates were washed five times in lysis buffer and analyzed by immunoblotting. For detection of CRACR2A-a, 5 × 10⁶ HEK293 or CRACR2A-deficient Jurkat T cells stably expressing empty vector, FLAG-tagged WT CRACR2A-a, CRACR2A^E278D, or CRACR2A^{R144G, E300*} were lysed in in RIPA buffer (10 mM Tris-Cl pH 8.0, 1% Triton X-100, 0.1% SDS, 140 mM NaCl, 1 mM EDTA, 0.1% sodium deoxycholate and protease inhibitor cocktail [Roche]) and centrifuged to remove debris. For immunoblot analyses, lysates were separated on 10% SDS-PAGE and proteins were transferred to nitrocellulose membranes and subsequently analyzed by immunoblotting with relevant antibodies. Chemiluminescence images were acquired using an Image reader LAS-3000 LCD camera (FujiFilm).

Cells were lysed in RIPA lysis buffer (50 mM Tris pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 150 mM NaCl, 1 mM Na3VO4, 20 mM NaF, 1mM PMSF, 2 mg ml⁻¹ aprotinin, 2 mg ml⁻¹ leupeptin and 0.7 mg ml⁻¹ pepstatin) for 1 h with intermittent vortexing and centrifuged to remove debris. Samples were separated on 10% SDS-PAGE. Proteins were transferred to nitrocellulose membranes and subsequently analyzed by immunoblotting with relevant antibodies. Chemiluminescence images were acquired using an Image reader LAS-3000 LCD camera (FujiFilm).