Lightning plus ecl reagent

Tissues were lysed in RIPA buffer as described in Müller et al., 2018 (link). Equal amounts of protein were separated by SDS-PAGE and transferred to a PDVF membrane. For protein detection, the following antibodies were used: C/EBPβ (E299, ab32358, 1:1000) from Abcam, C/EBPα (D56F10, #8178, 1:1000), PPARγ (C26H12, #2435, 1:1000), FAS (C20G5, #3180, 1:1000) and GAPDH (14C10, #2118, 1:1000) from Cell Signaling, SREBP1 2A4, MS-1207-PO, 1:1000 from NeoMarkers, and HRP-linked anti rabbit or mouse IgG from GE Healthcare. For detection, Lightning Plus ECL reagent (Perkin Elmer) or ECL prime reagent (GE Healthcare) was used. For re-probing, the membranes were incubated for 15 min with Restore Western Blot Stripping buffer (Thermo Fisher). The Image Quant LAS Mini 400 Imager or the Image Quant 800 Imager (both GE Healthcare) were used for detection and quantification of C/EBPβ LAP and LIP isoforms was performed using the supplied software.

Mouse liver and WAT tissue was homogenized on ice with a glass douncer in RIPA buffer (150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM TRIS pH 8.0 supplemented with protease and phosphatase inhibitors). Liver extracts were sonicated immediately, WAT extracts were incubated for 1 hr on ice, centrifuged for 15 min at 4°C after which the lipid layer was carefully removed using a cotton bud and then sonicated. Equal amounts of total protein were separated by SDS-PAGE, transferred to a PVDF membrane and incubated with the following antibodies: C/EBPβ (E299) from Abcam, β-actin (ab16039) from Abcam or (# 69100, clone C4) from MP Biomedicals; 4E-BP1 (C-19) from Santa Cruz; phospho-p70S6K (Thr389) (108D2), p70S6K (#9202), phospho-S6 ribosomal protein (Ser235/236) (2F9), S6 ribosomal protein (54D2), and phospho-4E-BP1 (Thr 37/46) (#9459) from Cell Signaling Technology and HRP-linked anti rabbit or mouse IgG from GE Healthcare and HRP-linked anti goat IgG from Santa Cruz. Lightning Plus ECL reagent (Perkin Elmer) was used for detection and for re-probing membranes were incubated in Restore Western Blot Stripping buffer (Thermo Fisher). The detection and quantification of protein bands was performed with the Image Quant LAS 4000 Mini Imager (GE Healthcare) using the supplied software.