Wet tank blotting system

Adipocytes, skeletal muscle cells or cell pellets were resuspended in RIPA buffer containing protease inhibitor cocktail and 1mM PMSF, sonicated on ice and lysates were centrifuged for 10 min at 10,000g and protein concentrations of supernatant were determined following a previously described method [10 ]. Protein from tissue extract or cell lysates or media was resolved on 10% SDS-PAGE and then transferred to PVDF membranes (Millipore, Bedford, MA, USA) with the help of Wet/Tank Blotting System (Bio-Rad Laboratories Inc, Hercules, CA, USA). Membranes were probed with specific primary antibodies (1:1000) and then detected by using secondary antibody conjugated with alkaline phosphatise (1:3000). 5-bromro-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) was used for detection of the protein bands. Intensity of the bands was analyzed using Image Lab Software (Bio-Rad Gel DocTMXR+, USA). α-Tubulin was used as a loading control and it was not affected by incubation with insulin or dmp. Similarly, the amount of tubulin was not different among BL6, db/db and dmp treated db/db mice samples.

Western blots to confirm MBD3 knockdown were performed as previously described [22 (link)]. In brief, cells expressing shRNAs for 7 days were acquired by cell sorting, lysed and proteins resolved by SDS–PAGE. Blotting was performed by using a wet-tank blotting system (BioRad, Hercules, CA, USA), and membranes were stained with primary antibodies anti-MBD3 (1:5000), anti-histone H3 (1:2000) or anti-β-actin (1:5000). Membranes were stained with secondary antibodies IRDye^® 800CW (LI-COR Biosciences, Lincoln, NE, USA) or IRDye^® 680RD (LI-COR Biosciences, Lincoln, NE, USA) (1:15,000) and imaged by using the Odyssey Infrared Image System (LI-COR Biosciences, Lincoln, NE, USA).

For western blotting, cortical neurons were lysed with RIPA buffer (Sigma-Aldrich) with 0.1% SDS and protease inhibitor cocktail (Sigma-Aldrich). The protein lysates were heated at 56°C for 30 mins to inactivate rabies virus [54 (link)]. The protein concentration in the lysates were determined using DC protein assay kit (Bio-Rad). 30μg of lysates were mixed with NuPAGE LDS sample buffer (ThermoFisher), heated at 90°C for 5 mins and then loaded on to Bolt 4–12% Bis-Tris Plus Gels (ThermoFisher). The gels were run at 150 volts for 1hour in Bolt MOPS SDS running buffer (ThermoFisher). Proteins were then transferred onto PVDF membrane blots (Bio-Rad) using wet tank blotting system (Bio-Rad), blocked with 3% BSA in PBSA with 0.2% Tween 20 (Merck Millipore) and probed overnight at 4°C with the following concentrations of primary antibodies diluted in 1% BSA/PBSA: chicken anti-MAP2 (1:1000, ABCAM, cat#ab92434), mouse pan-axonal neurofilament antibody (1:1000, BioLegend, cat#837904) and mouse anti- β actin (1:2000, Sigma-Aldrich, cat#A2228). The blots were then washed with 0.5% Tween 20 in PBSA and incubated with appropriate species-specific fluorescent secondary antibodies (Alexa Fluor, ThermoFisher) for 1hour at room temperature. The blots were imaged using iBright FL1000 imaging system (ThermoFisher).