Rabbit anti mouse igg

Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, and 1% Triton X-100) containing 1× Protease Inhibitor Cocktail (Roche, Indianapolis, IN) and 1× Phosphatase Inhibitor (Beyotime). Supernatants were separated and incubated with anti-FLAG (F3165; Sigma), anti- OGT (ab96718, Abcam), anti-KAT5 (sc166323; Santa), or anti-c-Myc (10828-1-AP; Proteintech Group Inc.) overnight at 4 °C. Protein-antibody complexes were incubated with protein A/G agarose beads (Millipore) for 4 h. The complexes were eluted and resolved to immunoblotting with the indicated antibodies. The horseradish peroxidase-conjugated secondary antibody was goat anti-mouse IgG (ab6789; Abcam) or mouse anti-rabbit IgG, light chain specific (211-032-171; Jackson ImmunoResearch, Lancaster, USA).

Protein G-Sepharose was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Precast NuPAGE® Bis-Tris gels were from Thermo Fisher Scientific (Waltham, MA, USA). NE and AngII were bought from MedChemExpress (Shanghai, China). All other chemicals were from Sigma-Aldrich (Shanghai, China) or Sangon Biotech (Shanghai, China). The commercial primary antibodies used in this study are listed in Supplementary Table 1, and used at a dilution of 1:1000 for immunoblotting. The RalGAPα1, RalGAPα2 and RalGAPβ antibodies were described previously^{14 (link)}, and used at a dilution of 1:1000 for immunoblotting. The antibody recognizing pThr⁴⁸⁴-SERCA2a was as previously reported^{7 (link)}, and used at 1 µg/ml for immunoblotting. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Cat No. 111-035-003), mouse anti-rabbit IgG (211-002-171), goat anti-mouse IgG (Cat No. 115-035-003) and goat anti-mouse IgG (115-005-174) were from Jackson ImmunoResearch Labs, and used at a dilution of 1:5000. Plasmids for RalGAPα1, RalA and SERCA2a were reported previously^{7 (link),16 (link)}.

Protein samples were quantified by Bradford assay using PRO-Measure solution (Intron, #21011) and subjected to protein electrophoresis (SDS-PAGE). Gels were blotted by wet transfer using a Bio-Rad Power Pac at 350 V for 1 or 2 h. Membranes were blocked and incubated with primary antibodies for overnight at 4 ℃. After washing, the membranes were incubated with secondary antibodies overnight at 4 °C. Next, after a washing step, the protein bands were detected using an ECL kit (XLS025-0000, Cyanagen) on the ChemiDoc system (Fusion Solo, Vilber Lourmat).
The primary antibodies used were: β-actin [Rabbit polyclonal antibody (Rab Poly Ab), Santa Cruz, sc-130656], HK1, HK2, PKM2, PDH [Rab Poly Ab, Cell Signaling Technology (CST), #8337], eIF2α (Rab Poly Ab, CST, #9722), peIF2α [Rabbit monoclonal antibody (Rab mono Ab), CST, #3597], CHOP (Mouse Monoclonal antibody, Invitrogen, #MA1-250), ANP (Rab poly Ab, Abcam, ab14348), SPT1 (Rab poly Ab, ABclonal, A6750), and PGRMC1 (Rab mono Ab, CST, #13856). The secondary antibodies used were: Mouse anti-rabbit IgG (211-032-171, Jackson ImmunoResearch, 1:5000) and Goat anti-mouse IgG antibody (BS-0296G-HRP, Bioss, 1:5000).

Cells were collected with PBS-5mM EDTA. 5x10⁵ cells were incubated in 100μL of 5μg/mL mouse IgG or 33B7 mAb in DMEM + 1% FBS for 1-hour at 4°C. Cells were then washed in 200μL cold PBS, and incubated with 10μg/mL Rabbit-anti-Mouse IgG (Jackson ImmunoResearch Laboratories, Cat. 315-005-003) in DMEM + 1%FBS for 1-hour at 4°C. Cells were then washed in 200μL cold PBS, and incubated in 20μg/mL Goat-anti-Rabbit-Alexa 647 in DMEM + 1% FBS for 1-hour at 4°C (Jackson ImmunoResearch Laboratories, Cat. 111-605-045). Cells were washed in 200μL cold PBS, then re-suspended in 160μL PBS, and binding was measured using an Intellicyt Flow Cytometer (Intellicyt HTFC Screening System).