Mouse anti erk1 2

Recombinant human APRIL was purchased from ProSpec-TanyTechnoGene (East Brunswick, NJ, USA). Antibodies against APRIL (ab64967), BCMA (ab5972), and TACI (ab64967) were purchased from Abcam (Cambridge, MA, USA). Rabbit anti-ERK1/2, mouse anti-ERK1/2 and rabbit anti-phospho-Akt S473 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Other reagents were from Sigma-Aldrich (St. Louis, MO, USA), except where specifically indicated. The lung cancer tissue microarray was obtained from Shanghai Zhuoli Biotechnology Co., Ltd. (Shanghai, China).

Cells were seeded at 4 × 10⁵ per well in six-well plates with appropriate growth medium and incubated at 37 °C, 5% CO₂ overnight, or treated with drugs for indicated period of time before harvesting. Cell lysates were prepared by cell lysis on ice with 1X RIPA buffer (Cell Signaling, #9806) containing protease inhibitor (Roche, #11836170001) and phosphatase inhibitor cocktail (Sigma, #P5726). Immunoblotting of individual protein bands was performed by incubating the PVDF membranes with the following primary antibodies (all purchased from Cell Signaling) diluted 1:1000 in 5% BSA/TBST: rabbit anti-phospho-ERK (#9101), mouse anti-ERK1/2 (#9107), rabbit anti-phospho-AKT (Ser473) (#4060), mouse anti-AKT (#2920), rabbit anti-phospho-FAK (Y397) (#8556), rabbit anti-FAK (#13009), and rabbit anti-GAPDH (#2118). Fluorescent Alexa 488-conjugated donkey anti-rabbit or anti-mouse antibodies (Life Technologies, A21206 or A21202) were used as secondary antibodies diluted 1:5000 in 5% BSA/TBST to visualize the protein bands on the Pharos Molecular Imager (BioRad). Intensities of individual protein bands from the scanned images were measured using ImageJ after subtracting background. Phosphoprotein levels were then normalized to either GAPDH or total level of the corresponding protein. Uncropped images of immunoblots are shown in Supplementary Figures 7 to 11.

Epididymal white adipose tissue (eWAT) was immersed in 1% paraformaldehyde fixative for 2 h. After fixing, the tissues were wax-embedded (Cellwax Plus paraffin wax, melting point 54–57 °C) using the Citadel 2000 Tissue Processor (ThermoFisher). Sections were cut at 5 μm with 100 μm intervals and dried at 37 °C for 48 h and subsequently stained with H&E (VWR) or immunofluorescent with ERK1/2 antibody mouse anti-ERK1/2 (4696 Cell Signaling Technology) and anti-mouse Alexa Fluor 488 (A11029 ThermoFisher). Tissues were imaged using a light microscope or a laser scanning confocal microscope (Zeiss). All analyses were done using Image J software blinded for treatment or genotype. Adipocyte size was manually measured in 500 adipocytes per animal and intensity of immunofluorescent staining was measured in 20 adipocytes per animal. Liver samples were snap-frozen in liquid nitrogen and cryosections (10 μm, with 50 μm intervals) were stained with 0.2% Oil Red O staining (Sigma-Aldrich), followed by haematoxylin essentially as in^{21 (link)}. Oil Red O staining was analysed 6 liver sections per animal. Images of en face longitudinal opened thoracic aortae were analysed for presence and coverage of plaque area as in^{22 (link)}.

Western blotting was performed in accordance with a previously described protocol (Linnskog et al., 2014). Briefly, the blotting assays were conducted using primary goat anti‐WNT5A (R&D Systems; 1 : 100), mouse anti‐ERK1/2 (Cell Signalling, Danvers, MA, USA, 1 : 1000), rabbit anti‐phospho‐ERK1/2 (Cell Signalling, 1 : 1000), rabbit anti‐EGFR (Cell Signalling, 1 : 1000), rabbit anti‐PDGFRβ (Cell Signalling, 1 : 1000) and mouse anti‐β‐actin (Sigma‐Aldrich, Stockholm, Sweden; 1 : 30 000) antibodies and secondary HRP‐conjugated rabbit anti‐goat, goat anti‐mouse or goat anti‐rabbit antibodies (Dako, Santa Clara, CA, USA; 1 : 10 000). MDA‐MB‐468 (MDA‐468) breast cancer cell lysates with or without recombinant WNT5A (rW5A) were used as positive and negative controls for WNT5A expression experiments. The densitometric quantifications of relative protein expression were conducted using image lab software (version 6.0, Bio‐Rad, Hercules, CA, USA).