Hybond lfp pvdf membrane

The western blot analysis was done as previously described previouusly (Choudhury et al., 2016 (link)). Briefly, VNC from wandering third instar larvae were dissected out in ice-cold HL3 buffer and homogenized in buffer containing 50 mm Tris-HCl, pH 6.8, 25 mm KCl, 2 mm EDTA, 0.3 m sucrose, and 2% SDS in water. The homogenized sample was then mixed with an equal volume of 2× Laemmli buffer. The protein equivalent to 50 μg was separated on 12% SDS-PAGE and transferred to Hybond-LFP PVDF membrane (GE Healthcare, GE Healthcare Life Sciences). The membrane was then blocked with 5% skimmed milk for 1 h, followed by overnight incubation with anti-Rab11 (1:2000) and anti-Tubulin (1:5000) antibody. IRDye 800 (1:10 000) was used as a secondary antibody, and signals were visualized on LI-COR Odyssey platform. The density of Western bands was quantified using ImageJ/Fiji software.

Dilutions of serum samples were made in Phosphate Buffered Saline (PBS), pH 7.2 and the equivalent of 0.75 μl of serum was separated by 10% SDS-PAGE under reducing conditions. Proteins were blotted onto Hybond-LFP PVDF membrane (GE Healthcare, Amersham, UK). Blots were blocked for 1 h in 5% w/v non-fat dried milk in PBS. Membranes were incubated for 2 h at room temperature with 1 μg/ml mouse monoclonal antibody to C1 esterase inhibitor (Abcam plc, Cambridge, UK), at a 1:2000 dilution. Blots were washed six times for 10 min with 0.1% Tween 20 in PBS before being incubated for 1 h with anti-mouse HRP labeled secondary antibody 1:2500 dilution. Blots were washed and dried and subjected to ECL (GE Healthcare, Amersham UK).

mRNA expression of Hdac7 and Tcf7l2 was analysed using TaqMan assays and related to expression of Ppia (Life Technologies) by quantitative real-time (q)PCR and the ΔΔC_t method. To verify overexpression of HDAC7 protein, clonal beta cells were transfected with haemagglutinin-tagged cDNA for Hdac7 and lysed in RIPA buffer (50 mmol/l Tris, pH 7.6, 150 mmol/l NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X100, protease inhibitor cocktail; Sigma-Aldrich, St Louis, MO, USA), and boiled with sample buffer (60 mmol/l Tris, pH 6.8, 10% glycerol, 2% SDS, 10% β-mercaptoethanol, bromophenol blue). Samples were separated on Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) and transferred onto Hybond-LFP PVDF membranes (GE Healthcare, Piscataway, NJ, USA). Protein expression was detected using a rabbit haemagglutinin tag (Abcam, Cambridge, UK; diluted 1:4000) and mouse β-actin (Sigma-Aldrich; diluted 1:10,000) antibodies, and secondary DyLight 680/800 conjugated goat antibodies (Thermo Scientific, Rockford, IL, USA; diluted 1:15,000), all validated by the respective suppliers. Blots were scanned using an Odyssey imaging system (LI-COR, Lincoln, NE, USA).