Anti rabbit igg antibody

Thapsigargin (TG), melatonin, rabbit polyclonal anti-β-actin antibody (catalog number A2066, epitope: amino acids 365–375 of human β-actin), bovine serum albumin (BSA), hydrocortisone (catalog number: H0888), insulin (catalog number: I9278), epidermal growth factor (catalog number: E9644), OAG and cholera toxin (catalog number: C8052) were from Sigma. Fura-2 acetoxymethyl ester (fura-2/AM) was from Molecular Probes. Rabbit polyclonal anti-TRPC6 antibody (catalog number: ACC-120, epitope corresponding to amino acid residues 573–586) was from Alomone. Rabbit polyclonal antiubiquitin antibody (catalog number: ab19247) was from Abcam. DharmaFECT kb transfection reagent was from Cultek. Mouse monoclonal anti-PMCA antibody (Clone 5F10, epitope: amino acids 724–783 of human PMCA) and Live/Dead viability/cytotoxicity kit were from Thermo Fisher. Horseradish-peroxidase-conjugated anti-mouse IgG antibody and anti-rabbit IgG antibody were from Jackson ImmunoResearch Europe Ltd. RNA control vector was from Origene. Enhanced chemiluminescence detection reagents were from Pierce. Bromodeoxyuridine (BrdU) cell proliferation assay kit was from BioVision. SAR7334 was from Quimigen. hTRPC6-YFP was a gift from Craig Montell (Addgene plasmid#21084; http://n2t.net/addgene:21084; RRID:Addgene_21084). All other reagents were of analytical grade.

Primary antibodies including anti-phospho-mTOR (Ser2448), anti-mTOR, anti-phospho-4EBP1 (Ser65), anti-4EBP1, anti-phospho-Akt (Ser473), anti-phospho-Akt (Thr308), anti-Akt, anti-phospho-rpS6 (Ser240/244), anti-phospho-rpS6 (Ser235/236), anti-rpS6, anti-p-p53 (Ser15), p53, anti-phospho-IGF-1Rβ (T1135/36/1150/51), IGF-1Rβ and anti-MYC antibodies were purchased from Cell Signaling Technology (Danfoss, MA, USA). The anti-Rabbit IgG antibody was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Anti-S6K1 antibody was purchased from Abcam (Cambridge, UK). Anti-β-actin, anti-FLAG (M2) and anti-p62/SQSTM1 antibodies were purchased from Sigma (Saint Louis, MO, USA), anti-LC3 antibody was purchased from Medical & Biological Laboratories (Nagoya, Aichi, Japan). Secondary antibodies used in western blotting including IRDye 800CW Donkey anti-Mouse and anti-Rabbit IgG were purchased from Li-COR (Lincoln, NE, USA). Secondary antibodies used in immunofluorescence including AlexaFluor-555-conjugated goat anti-mouse and AlexaFluor-555-conjugated goat anti-rabbit antibodies were purchased from Invitrogen (Burlingame, CA, USA).

Cell lysates were collected in TNE-N+ buffer (150 mM NaCl, 20 mM Tris-HCl, pH 8.0, 1 mM EDTA, and 1% NP-40) in the presence of various inhibitors. To detect the indicated proteins, anti-DJ-1 antibody 3E8 (MBL, 1:1000), anti-Tubulin antibody YL1/2 (Abcam, 1:3000), anti-HA antibody TANA2 (MBL, 1:2000), anti-TOMM40 antibody (a gift from Dr Toshihiko Oka, Department of Life Science, Rikkyo University, Japan, 1:1000), anti-TIMM23 antibody (BD Biosciences, 1:500), anti-TOMM20 antibody (Proteintech, 1:4000), anti-OPA1 antibody (BD Biosciences, 1:1000), anti-PGAM5 antibody (Abcam, 1:1000), anti-ATF4 antibody (CST, 1:500), anti-Actin antibody C4 (Merck Millipore, 1:2000) or an anti-GFP antibody ab6556 (Abcam, 1:2000) were used as primary antibodies, and HRP-conjugated goat anti-mouse IgG or anti-rabbit IgG antibody was used as secondary antibodies (Jackson ImmunoResearch). The HRP substrate consisted of the Western Lightning Plus-ECL (PerkinElmer) and images were captured on an ImageQuant LAS4000 (GE Healthcare). Quantification was performed using Image J software. Antibodies used in this study and their Research Resource Identifiers (RRIDs) are listed in Table S1. To provide cellular stress in Fig. S2, WT HeLa cells were treated with DMSO (NT, non-treated), 10 µM valinomycin (val) or 300 nM thapsigargin (TG) for 3 h, and then total cell lysates were prepared for immunoblotting.

After corresponding animal experiment, rats were sacrificed via cervical dislocation. Knee-joint was extracted and fixed in 4% paraformaldehyde. Then tissues were dehydrated in ethanol and finally embedded in paraffin. 5 mm-thick histologic cuts from the paraffin blocks were obtained and stained with hematoxylin-eosin (HE) for general histology. Immunohistochemistry for Orai1 (1:50; Ab59330, Abcam), was performed after antigen retrievals in citrate buffer. Samples were incubated with a secondary antibody (anti-rabbit IgG antibody, Jackson ImmunoResearch) and mounted with mounting media. Orai1 was stained as brown particles. The images of the stained tissue were captured via a light microscope.

Samples were collected using 1 × SDS buffer and run on 10% polyacrylamide gels. Gels were transferred in the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked using 5% BSA in 1× tris-buffered saline with 0.2% Tween (TBST) and probed with primary eIF4G (1:1,000; Santa Cruz Biotech), ODC1 (1:1,000; Invitrogen), SAT1 (1:1,000; Novus Biologicals), and b-actin (1:1,000; ProteinTech) antibody. Membranes were washed in 1× TBST and placed in secondary anti-mouse IgG (1:15,000; Jackson Immunoresearch) or anti-rabbit IgG antibody (1:15,000; Jackson Immunoresearch) and incubated at room temperature for 1h. Again, membranes were washed in 1× TBST, and SuperSignal West Pico Plus chemiluminescent substrate (Thermo-Fisher) was applied to membranes and developed on a molecular imager, Bio-Rad GelDoc XR1 imaging system (Bio-Rad). Images were quantified with ImageJ.