6xhis

Human lung epithelial cell line A549 (ATCC), its hnRNP E1 knockdown (A549/E1KD or E1KD), normal mouse mammary epithelial (NMuMG) cell line, and its hnRNP E1 knockdown (NMuMG/E1KD or E1KD) derivative cells (Hussey et al, 2011 (link), 2012 (link)) were cultured in DMEM supplemented with 5% FBS and 5% FCS at 37°C in a 5% CO₂ humidified chamber. Antibodies used in the work were against the following proteins: hnRNP E1 (mouse; Abnova; mouse, MBL; and rabbit, raised against purified hnRNP E1), TRF2 (rabbit, NB10056506; Novus), Hsp90 (mouse; SCBT), FLAG (rabbit; Sigma-Aldrich), 6XHis (rabbit; Cell signaling), phospho-ATM (MAB 2401; Abnova), ATM (2873S; Cell signaling), phospho-ATR, ATR (13934S; Cell signaling), phospho-p53 (9284S; Cell signaling), p53 (2524; Cell signaling), γ-H2A.X (rabbit, 9718S; Cell signaling), Histone H2A.X (7631T; Cell signaling), PCNA (mouse, Cat no. 2586S; SCBT), Usp1 (rabbit, 8033S; Cell signaling), Usp7 (mouse, HAUSP; SCBT), RPA32 (rat, 2208S; Cell signaling), Rad6 (R6A/R6B, rabbit, 4944S; Cell signaling), Rad18 (rabbit, 9040S; Cell signaling), and Fen1 (rabbit; SCBT). Plasmids containing hnRNP E1 and its three KH domains namely KH1, KH2, and KH3 have been described previously (Chaudhury et al, 2010b (link); Brown et al, 2016 (link)); each of the four ORFs contained an N-terminal GST tag.

GST, 4G10 and phospho-^(S79)-ACC, antibodies were from Millipore (Billerica, MA, USA), 6xHis, phospho^-(T172)-AMPK, total-AMPK and total-ACC antibodies were from Cell Signaling (Danvers, MA,USA), LKB1 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA,USA) and Millipore (Billerica, MA,USA), GAPDH and tubulin alpha antibodies were from Abcam (Cambridge, MA, USA). Flag antibody was from Sigma-Aldrich (St. Louis, MO, USA), and the V5-epitope antibody was from MBL international (Woburn, MA, USA). Phospho LKB1 tyrosine 261 and 365 specific antibodies (pY261- and pY365-LKB1) were developed in our laboratory [17] (link). GST-Fyn fusion protein was purchased from Millipore (Billerica, MA, USA). TAT-LKB1 modified peptides (11R-WT and 11R-Sca) were synthesized by BIOMATIK (Wilmington, DE, USA). pcDNA3-Fyn-WT, pcDNA3-Flag-LKB1-WT and pcDNA3-Flag-LKB1-Y261/365F were constructed and described before [17] (link). pGEX was a generous gift from Dr. Bridget Shafit-Zagardo (Albert Einstein College of Medicine, NY,USA). C2C12 muscle cells were obtained from American Type Culture Collection (ATCC) (Manassas, VA, USA).