Anti ha tag antibody

The protein extraction and protein gel blot assay were done as described [27] (link). Anti-HA tag antibody (Roche), Anti-RBS1 antibody(Beijing Protein Innovation, China), Anti-SPIN1 antibody(Beijing Protein Innovation, China) and Anti-peroxidase (PAP) antibody (Sigma-Aldrich) were used to detect these proteins. Chemiluminescene was detected by the ChemiDoc XRS system (Bio-Rad).

Antibodies against phospho-eNOS (Ser1177), phospho-AMPKα (Thr172), AMPKα, phospho-Akt (Ser473), phospho-p38 mitogen-activated protein kinase (p38 MAPK) (Thr180/Tyr182), p38 MAPK and cAMP response element binding protein (CREB) were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against phospho-CREB (Ser133) and eNOS (NOS3) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-phospho-acetyl CoA carboxylase (ACC) (Ser79) antibody was purchased from Millipore (Billerica, MA). An anti-myc tag antibody was purchased from Upstate Biotechnology (Lake Placid, NY) and an anti-HA tag antibody was purchased from Roche (Basel, Switzerland).
Wortmannin, LY294002, and KT5720 were purchased from Cayman Chemical Company (Ann Arbor, MI). H89 and GSK3787 were purchased from Tocris Bioscience (Bristol, UK). GW9662 was purchased from Wako Pure Chemical (Osaka, Japan). SB202190 was purchased from Calbiochem (Darmstadt, Germany).
Valsartan and irbesartan were purchased from Vijayasri Chemicals (Andhra Pradesh, India). Telmisartan was provided by Boehringer Ingelheim (Ingelheim, Germany).

HeLa cells were grown to 70% confluency in DMEM + 10% FCS + 1% P/S and transfected with murine LIMP-2-myc for 48 h. Transfected and untransfected HeLa cells, WT MEFs and LIMP-2^−/− MEFs, WT MDCK cells were harvested, lysed, and analyzed with the Native PAGE™ Bis-Tris Gel System (ThermoFisher) according to the manufacturer’s instructions, or via SDS–PAGE, and immunoblotting. For co-immunoprecipication, HeLa cells were transiently transfected with GFP or co-transfected with two differently tagged LIMP-2 constructs (LIMP-2-myc and LIMP-2-HA) for 48 h. Cells were harvested, lysed and the lysate was incubated with anti-myc antibody (9B11, Cell Signaling) overnight at 4 °C. Then, magnetic agarose G beads (ThermoFisher) previously blocked with 3% BSA were added to the samples (40 min, 4 °C, rotating) for the precipitation of LIMP-2-myc. Afterwards, samples were analyzed via SDS–PAGE with reducing reagents and immunoblotting. An anti-HA-tag antibody (3F10, Roche) was used to detect co-precipitated LIMP-2-HA.