Mouse anti ha

Yeast cells expressing the indicated fusion proteins were harvested by filtration, frozen in liquid nitrogen, and cryogenically disrupted using the Precellys homogenizer in 4 mL of lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% NP-40, 10% glycerol, 400 mM Pefabloc, and Roche complete protease inhibitor EDTA-free) with the addition of 60 mM glutamine as in Ukai et al., 2018 (link). Cleared lysates were equilibrated in the same lysis buffer. For input samples, aliquots of cleared lysates were collected and denatured in presence of SDS-PAGE sample buffer. For co-immunoprecipitations, the cleared lysates were incubated for 2 h at 4 °C with prewashed anti-c-myc MagBeads (Pierce Thermo Fisher Scientific, product number 88843). After five washes with lysis buffer, beads were resuspended in 20 µL lysis buffer and denatured in presence of SDS-PAGE sample buffer. Inputs and pull-down samples were analyzed by SDS-PAGE immunoblot with mouse anti-myc (Santa Cruz, 1:10,000) and mouse anti-HA (ENZO, 1:1000) antibodies.

The following antibodies were used for western blotting (WB) and immunofluorescence (IF): mouse anti-beta-actin (Santa Cruz, catalog number sc-47778; WB 1:2000), mouse anti-HA (ENZO, catalog number ENZ-ABS120-0200; WB 1:1000), mouse anti-transferrin receptor (Invitrogen, catalog number 13–6800; WB 1:1000), rabbit anti-Stim1 (Cell Signaling Technology, catalog number 5668S (D88E10); WB 1:2000), rabbit anti-Orai1 (Sigma Aldrich, catalog number O8264; WB 1:2500), goat anti-myc tag (Abcam, catalog number ab9132; IF 1:2000), rabbit anti-RHBDL2 (Proteintech, catalog number 12467-1-AP; WB 1:250 – only detected RHBDL2 in HaCaT lysates), rabbit anti-V5 tag (Cell Signaling Technology, catalog number 13202S; WB and IF 1:2000). Corresponding species-specific HRP or fluorescently coupled secondary antibodies were used from Santa Cruz and Cell Signaling (WB) or Invitrogen (IF).

The following antibodies were used: mouse anti-beta-actin (Santa Cruz, catalogue number sc-47778; WB 1:2000), rabbit anti-TACE/ADAM17 (Abcam, catalogue number ab39161; WB 1:2000), rabbit anti-HA (Santa-Cruz, catalogue number sc-805; IF 1:1000), mouse anti-HA (ENZO, catalogue number ENZ-ABS120-0200; PM IP 1:1000), mouse anti-transferrin receptor (Invitrogen, catalogue number 13–6800; 1:1000), rabbit anti-phosphoserine (Invitrogen, catalogue number 618100; WB 1:1000), rat HA-HRP, clone 3F10 (Roche, catalogue number 12013819001; WB 1:2000), mouse anti-p97 (Pierce/Thermo, catalogue number MA1-21412; WB 1:1000), rabbit anti-pan14-3-3 (Cell Signalling, catalogue number 8312; WB 1:1000), mouse anti-GM130 (BD Transduction labs, catalogue number 610823; IF 1:1000), rabbit anti-pan-cadherin (Cell Signalling, catalogue number 4068S; WB 1:1000), rabbit anti-ADAM10 (Abcam, catalogue number ab1997; WB 1:1000), rabbit anti-ERK1/2 (Cell Signalling, catalogue number 9102, WB 1:1000), rabbit anti-pERK1/2 (Cell Signalling, catalogue number 4377, WB 1:1000). Corresponding species-specific HRP or fluorescently coupled secondary antibodies were used from Santa Cruz and Cell Signaling (WB) or Invitrogen (IF).