Hrp conjugated secondary igg

Spheroids with and without treatments (24 h) were lysed in RIPA buffer containing Na₃VO₄ (1 mM) and protease inhibitors (1 μg/ml). For subcellular fractionation, spheroids (in 24-well plates) were treated with GW-exo-miR for 24 h, harvested, and the pellet re-suspended in the digitonin cell permeabilization buffer (Trevigen, Gaithersburg, MD). The supernatant containing the cytosolic fraction was collected. The remaining pellet (organelle fraction) was lysed in the RIPA buffer containing Na₃VO₄ (1 mM) and protease inhibitors (1 μg/ml). Protein concentration was assessed by the Bradford assay. The lysates (10 μg of protein) were separated using 4–12% SDS-PAGE (Life Technologies) and transferred onto a nitrocellulose membrane (Protran). After blocking with 5% non-fat milk in PBS-Tween (0.1%), the membranes were incubated overnight at 4  °C with primary antibodies against LC3B, phospho-mTOR (p-mTOR^ser-2448), mTOR, phospho-p70S6K^ser-235/236, p70S6K, PARP-1, BID, BAX, Caspase-8, Caspase-9, Caspase-3, cyt-c, AIF, pAMPK^thr-172, AMPK, pULK1^ser-555 and ULK1 (all Cell signaling). β-Actin or GAPDH (Cell Signalling) was used as a loading control. After incubation with HRP-conjugated secondary IgG (Cell Signalling), the blots were developed using ECL (Pierce). The band intensities were visualized and quantified with ChemiDoc using Quantity One software (Bio-Rad Laboratories).

The snap-frozen brain tissue samples were homogenized in 1 ml Buffer A (10 mM Tris-HCl pH = 8, 250 mM Sucrose, 10 mM MgCl2, 1 mM EGTA) with 1X protease inhibitor to extract cytoplasmic proteins. The resulting pellets were further homogenized in 1 ml RIPA buffer containing 1X protease inhibitor to isolate nuclear fraction. The total protein concentrations in cytoplasmic and nuclear fractions were quantified by Bradford assay (Bio-Rad). Bulk chromatin extraction was performed with histone extraction kit from Active Motif. For western blot, equal amounts of protein from normal or GBM specimens were denatured in final 1X SDS stop buffer and subjected to SDS-PAGE for western blot analysis with antibodies against H3K4me3 (Millipore #07-473; 1:1000), H3, H4, MLL1, MLL2, MLL3, and γ-tubulin (Sigma #T5326; 1:1000). Subsequently, HRP-conjugated secondary IgG (Cell Signalling; 1:2000) and enhanced chemiluminescence kit (ECL plus; GE) were used for detection. Of note, blotting membranes were cut into strips to accommodate the molecular weights of MLL, H3K4me3, and γ-tubulin for antibody incubation separately. Anti-H3K4me3 was stripped after ECL detection and the blots were subsequently incubated with unmodified H3 and H4 antibodies (internal controls).