Anti calnexin

The pharmacological inhibitors BIX02189 and WNK463 were obtained from Selleckchem (Houston, TX, USA) and trametinib from LC Laboratories (Woburn, MA, USA). General chemical reagents were purchased from Sigma–Aldrich (St Louis, MO, USA), Merck (Darmstadt, Germany) or BD Biosciences (San Jose, CA, USA).
The antibodies were obtained as follows: anti‐MEK5 was from Enzo Life Sciences (Farmingdale, NY, USA); anti‐GAPDH and anti‐ERK1/2 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti‐pERK1/2, anti‐MEK1/2, anti‐pMEK1/2, anti‐WNK1 and anti‐MEKK2 from Cell Signaling Technologies (Danvers, MA, USA); anti‐Calnexin from Stressgen Bioreagents (Victoria, BC, Canada); anti‐Ki‐67 (clone SP6) from Vitro Master Diagnostica (Granada, Spain); anti‐pWNK1 from R&D Systems (Minneapolis, MN, USA); and horseradish peroxidase‐conjugated secondary antibodies were from Bio‐Rad Laboratories (Hercules, CA, USA). Antibodies raised against ERK5, pERK5 or pMEK5 were developed in our laboratory and have been described.¹⁹, ²⁵, ²⁶ The pERK1/2 antibody conjugated to PE fluorophore was obtained from Biolegend (San Diego, CA, USA) and the anti‐pERK5 conjugated to AF488 from Santa Cruz Biotechnology (p‐ERK 5 Antibody (1.T218/Y220) Alexa Fluor^®488, sc‐135760 AF488).

All cells were maintained in a 37 °C, 5% CO₂ humidified incubator in corresponding medium, namely, minimum essential medium for HEK293 WT (HEK) and stably expressing Orai1 (HEKO1), AMIV for human CD4+ cells and RPMI 1640 for E6.1 Jurkat T (Jurkat) cells. All media were supplemented with 10% fetal calf serum and 1% penicillin–streptomycin and HEKO1 were maintained in 1 μg ml⁻¹ puromycin. HEK cells were passaged by treatment with trypsin/EDTA. For transfection, the indicated amount of DNA was electroporated into HEK cells and Jurkat cells with Nucleofector II or into CD4+ cells with 4D Nucleofector core unit according to the manufacturer's instructions 16–20 h before measurements. Human Orai1, Orai2 and STIM1 were subcloned into the EcoRV site of either RFP pMAX, or IRES-RFP-pCAGGS. STIM2.2 was purchased with an N-terminal CFP or YFP tag (Addgene). We used this cDNA (STIM2.2: pEX-CMV-SP-YFP-STIM2) and two complementary primers (see Supplementary Table 1) each encoding part of the exon 9 sequence to insert the desired nucleotides into the exon 8/10 boundary. All constructs were confirmed by sequencing. Antibodies used in this study were anti-His Tag (Cell Signaling, 2366, 1:1,000); anti-STIM2 (C-term, Sigma, #S8572, 1:2,000); anti-Calnexin (Stressgen, #SPA-865, 1:1,000) and anti-γ-tubulin (Cell Signaling, #5886, 1:1,000).

Cell culture media, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hematoxylin and eosin were purchased from Sigma. Foetal bovine serum (FBS) and antibiotics were from Life Technologies and Immobilon P (PVDF) membranes from Millipore. OOS was provided by Catalysis, S.L. (Madrid, Spain). The Cytometric Bead Array Mouse Inflammation kit was purchased to BD biosciences. Other generic chemicals were purchased from Sigma Chemical Co., Roche Biochemicals or Merck.
The origin of the different antibodies used in the Western blotting analyses were as follows: the anti-GAPDH, anti-p21, anti-Wee1, anti-CDC2/CDK1, anti-pCDC2, anti-Cyclin B and anti-Cyclin D1 antibodies were purchased from Santa Cruz Biotechnology; the anti-BUBR1, anti-Rb, anti-Cyclin A, anti-Cyclin D3 and anti-Cyclin E from BD Biosciences; the anti-p27 and the anti-pRb^S780 from Cell Signaling technology; the anti-Calnexin from Stressgen and the anti-pHistone H3 from Millipore. The horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad.