Anti tg2

Western blotting analysis was carried out as previously reported [32 (link)]. Whole cell extracts (10 μg), obtained using CelLytic™ MT Cell Lysis Reagent (Merck Life Science, Cat.No. C3228), were separated on SDS-PAGE 10% gels and electroblotted onto nitrocellulose membranes (Merck Life Science, Cat.No. GE10600041). Blots were incubated with primary antibodies in 5% nonfat dry milk (Biosigma, Venezia, Italy, Cat.No. 711160) in PBS (Thermo Fisher Scientific, Waltham, MA, USA, Cat.No. J61196.AP) plus 0.1% Tween-20 (Merck Life Science, Cat.No. P1379) overnight at 4 °C. The primary antibodies used in this study were anti-TG2 (Thermo Fisher Scientific Cat.No. MA5-12739) and anti-actin (Merck Life Science, Cat.No. A-2066). Detection was achieved using horseradish peroxidase-conjugated secondary antibodies (anti-mouse 715-036-150 or anti-rabbit 711-036-152; Jackson ImmunoResearch Laboratories, Ely, UK) and enhanced chemiluminesence (ECL) [Immobilon Classico Cat.No. WBLUC0500 and Immobilon Crescendo Western HRP substrate Cat.No. WBLUR0500 Merck Life Science]. Signals were acquired using a ChemiDoc imaging system (Bio-Rad, Milan, Italy).

The following antibodies were used: anti-TG2 (Thermo Scientific, CUB7402), anti-PKM2 (Cell Signalling, D78A4), anti-EEF1A1 (Milipore, 05-235), anti-Hsc70 (Abcam, 51052), HSPA1A (Santa Cruz, 7947), anti-Biotin (Abcam, 1227), anti-p62/SQSTM1 (mbl, PM045), anti-LC3 (Novus Biologicals, NB100-2331), anti-GAPDH (Sigma, G9545), anti-Actin (Sigma, 2066), anti-Beclin1 (Santa Cruz, 10086), anti-HIF1-beta (Novus Biologicals, NB100-124), anti-ULK1(Santa Cruz, 33182), anti-phospho-tyrosine (Cell Signaling, 9411) and HRP-conjugated secondary antibodies (Bio-Rad Laboratories).