Anti rabbit igg hrp na934v

To generate peptide arrays, human lamin A (P02545, full length), human lamin C (NP_005563, full length), mouse lamin A (P48678, 588-end), and rat lamin A (P48679, 588-end) were synthesized as 20 mer peptides with three amino acid offsets on cellulose membranes using a Multipep automated peptide synthesizer (INTAVIS Bioanalytical Instruments AG, Koeln, Germany) as described [30 (link)]. Peptide array membranes were blocked for 2 h in 1% casein in TBST (Tris-buffered saline with 1% tween) at 22–25 °C, and then incubated overnight at 4°C with a primary antibody (5G4 at 1:2500 dilution; L1283 at 1:2000) in TBST/1% casein. Membranes were then washed three times in TBST (10 min each) and incubated with affinity-purified horseradish-peroxidase-conjugated polyclonal anti-mouse IgG HRP (NA931V) or anti-rabbit IgG HRP (NA934V), both from GE HealthCare (Little Chalfont, UK). Blots were developed using ECL Prime (RPN 2232, GE HealthCare) and chemiluminescence signals were detected using Las 1000 (Fujifilm, Tokyo, Japan). Lasergene software (DNAStar, Madison, WI, USA) was used to generate peptide sequence alignments.

The cellular proteins were extracted using RIPA buffer and protein concentrations were measured by BCA protein assay kit. Triplicate protein samples were run in 4–12% NuPAGE Bis-Tris gels (Life Technologies, Espoo, Finland) and transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Uppsala, Sweden). Membranes were blocked in 3–5% milk or BSA in TBS-Tween-20, washed with TBS-Tween-20 and incubated overnight at +4 °C with primary antibodies followed by washing with TBS-Tween-20 and incubation with secondary antibodies for 1 h at room temperature. The bands were visualized using enhanced chemiluminescence and images were captured with Image Quant RT-ECL system (GE Healthcare). Bands were quantified by Quantity One software (Bio-Rad, Hercules, CA, USA). The antibodies were from following sources: SIRT1 (07–131) was from Millipore. Phospho-AMPK (2535), AMPK (2532) and FOXO1 (2880) were from Cell Signaling Technology, Beverly, MA, USA. PGC1α (sc-13067), Ac-FKHR (acetylated FOXO1, sc-49437), NRF1 (sc-13031), GLUT4 (sc-1608) and donkey anti-goat IgG-HRP (sc-2020) were purchased from Santa Cruz. Anti-rabbit IgG-HRP (NA934V) and anti-mouse IgG-HRP (NA931V) were from GE Healthcare. α-tubulin (T5168) was from Sigma. The details of the antibody dilutions are shown in the Supplementary Table 1.