Sc 2955

Protein extraction and Western blotting were performed as described [30 (link)], by applying anti-rabbit antibodies against BMP8B (#PAB25185) from Abnova (Jhouzih St., Taipei, Taiwan; diluted 1:500) and phospho-IκBα (#2859) and phospho-NFκBα p65 (#3033) antibodies from Cell Signaling Technology (Danvers, MA, USA) (all diluted 1:1000). Furthermore, antibodies against actin (sc-2955, from Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:5000) or against DGAT2 (orb19428, from Biorbyt, Cambridgeshire, UK; 1:1000) were applied. For densitometry analysis, Licor Image Studio Lite software (Licor) was used. For (immuno)histochemical analysis, standard 5 µm sections of formalin-fixed and paraffin-embedded tissue blocks were stained using the BMP8B (#PAB25185) antibody (diluted 1:50).

To analyse protein phosphorylation, 3–5 million BMDCs or 2 million pMacs were stimulated with LPS or poly I:C as indicated and cells lysed in lysis buffer (150 mM NaCl, 50 mM TrisHCl pH8.0, 0.1% SDS, 0.5% Sodium Deoxycholate, 1% Triton) supplemented with protease (cOmplete, Sigma) and phosphatase (PhosSTOP, Sigma) inhibitors. Samples were resolved by SDS-PAGE and proteins transferred to PVDF membranes. The following antibodies were used for western blot (all from Cell Signalling unless specified otherwise): β-actin (1:3000, 8H10D10), ERK (1:1000, L34F12), p-ERK (1:1000, 9101), p38 (1:1000, D13E1), p-p38 (1:1000, 28B10), p65 (1:1000, D14E12), p-p65 (1:1000, 93H1), CD36 (1:1000, PA5-33291, ThermoFisher), anti-mouse IgG (1:3000, Poly4053, Biolegend), anti-rabbit IgG (1:3000, sc-2955, Santa Cruz Biotechnology), PPARδ (1:500, ab23673, Abcam). Membranes were imaged in an Amersham Imager 600 and band intensity quantified with Fiji (ImageJ).