Anti phospho ampkα thr172 40h9

BMDM were washed three times with 2 ml of ice-cold PBS and lysed with 0.15 ml of M-PER mammalian protein extraction reagent with Halt protease and phosphatase inhibitors (Thermo Scientific). Equal amounts of protein per sample/lane were denatured and resolved by SDS-PAGE. Proteins were transferred to Immobilon-FL membranes (Millipore, Billerica, MA), and quantitative immunoblotting was performed using the Odyssey infrared immunoblotting detection system (LI-COR Biosciences, Lincoln, NE). Primary antibodies used were anti-CaMKK2 (6/CaM Kinase, 610544; BD Biosciences; dilution 1:1000); anti-actin (AC-40, catalog # A4700; Sigma-Aldrich; dilution 1:20000); anti-phospho-CaMK1 (Thr¹⁷⁷; PA5-38434 ThermoFisher, dilution 1:500), anti-phospho-AMPKα (Thr¹⁷², 40H9; catalog # 2535; dilution 1:1000) and AMPKα (F6, catalog # 2793; dilution 1:1000) were from Cell Signaling (Danvers, MA). Secondary antibodies used were anti-mouse IgG Alexa Fluor 680 (catalog # A28183; Invitrogen; dilution 1:1000) and anti-rabbit IgG IRDye800-conjugated antibody (catalog # 611-132-002; Rockland Immunochemicals, Gilbertsville, PA; dilution 1:5000). All antibodies were used according to the manufacturer’s instructions. Images of uncut and unmanipulated blots are presented in Supplementary Fig. 12.

Human recombinant full-length adiponectin protein (composed of the LMW, MMW, and HMW isoforms) with a C-terminal FLAG sequence (produced in HEK293 cells, cat. no. RD172023100) and the globular form of adiponectin protein with an N-terminal His-tag sequence (produced in Escherichia coli, cat. no. RD172112100) were purchased from BioVendor (BioVendor, LLC, Asheville, NC). The following antibodies were used: anti-adiponectin polyclonal antibody (cat. no. RD181023100; BioVendor), anti-LOX-1 (#1-1) (17 (link)), anti-p44/42 MAPK (extracellular signal-regulated kinase [Erk] 1/2) (cat. no. 9102, Cell Signaling, Danvers, MA), anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (cat. no. 9101, Cell Signaling), anti-AMPKα (F6; cat. no. 2793, Cell Signaling), anti-phospho-AMPKα (Thr172) (40H9; cat. no. 2535, Cell Signaling), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (cat. no. NA934, GE Healthcare), donkey anti-chicken IgY (cat. no. AP194P, Millipore, Burlington, MA), sheep anti-apoB polyclonal antibody conjugated with HRP (cat. no. PP086, The Binding Site, Birmingham, UK), and anti-apoB chicken monoclonal antibody (HUC20) (18 (link), 19 (link)). HUC20 was developed as previously described (18 (link)). Epitope mapping showed that the HUC20 binding epitope of apoB is within the B1 region (amino acids 28–217 of apoB) (19 (link)).

Cell lysates of 293T transfected with FLAG-tagged constructs of PPM1A-wild type, PPM1A-R174G, PPM1F, and PPM1F-R326A-I328A were precleared with mouse IgG and A/G-agarose suspensions (Santa Cruz). Then the lysates were incubated with anti-FLAG antibody (M5) and protein A/G-agarose suspensions (Santa Cruz). Immune complexes bound to protein A/G-agarose were precipitated and incubated at 30°C for 30 min with recombinant active AMPK (α1β1γ2) (PV6238; Thermo Fisher Scientific) in 1× NEB buffer for PMP and 1 mM MnCl₂ (P0753S; New England Biolabs). Lambda Protein Phosphatase was used as a positive control (P0753S). The reaction mixtures were separated on 9% SDS-polyacrylamide gels and transferred to PVDF membranes. Phosphorylated AMPKα1 was immunodetected with anti-phospho-AMPKα Thr172 (40H9; Cell Signaling Technology).