Flag tag (flag)

For hypoxic treatment, HUVECs were incubated under either 20% O₂ or 1% O₂ for 24 h. The levels of HIF-1α, Siah2, pSiah2, PHD3, p38 and phospho-p38 were analyzed by Western blot using specific antibodies (Cell Signaling, Danvers, MA, USA). SB 203580 (Cell Signaling) at 10 μM was used to inhibit p38 phosphorylation.
For immunoprecipitation, HeLa cells were lysed with lysis buffer (Cell Signaling) and immunoprecipitated with antibodies against Flag (Sigma, St. Louis, MO, USA) or VEGFR1 (Genetex, Irvine, CA, USA) overnight, followed by protein A/G magnetic beads (Pierce, Waltham, MA, USA). The membranes were probed with antibodies against Vav1 (EMD Millipore, Billerica, MA, USA), Flag, HIF-1α (BD Biosciences, San Jose, CA, USA), VEGFR1 (Abcam, Cambridge, UK) and Ubiquitin (Cell Signaling). Western blot images were quantified by using densitometry [23 (link)].

mAbs for human CD3 (APC-H7; SK7; 560176), CD4 (BV711; SK3; 563028), CD8 (APC; SK1; 340584), CD45RA (PE; HI100; 555489), CD45RO (BV786; UCHL1; 564290), CD69 (FITC; L78; 347823), CCR7 (FITC; 150503; FAB197F-100), PD-1(PE-Cy7; EH12.1;561272), Lag3(PE;T47-530;565616), FLAG (APC; L5; 637308), Granzyme-B (PE;GB11;561142) from BD biosciences and BioLegend were used. Samples were acquired with BD FACSCanto II or BD LSRFortessa. A minimum of 10 000 events were acquired for each sample and were analyzed using FlowJo 10 (FlowJo).

The following reagents were used: NAC (Sigma-Aldrich, Cat# A7250), tunicamycin (Enzo Life Sciences, Cat# BML-CC104), glycine (VWR, Cat# 10119CU), tBHQ (Sigma-Aldrich, Cat# 19986), ML385 (Sigma-Aldrich, Cat# SML1833), Rapamycin (Sigma-Aldrich, Cat# R0395), Torin 2 (Selleckchem, Cat# S2817), serine (Sigma-Aldrich, Cat# S4500), asparagine (Sigma-Aldrich, Cat# A4159), VEGF-A 165 (R&D, Cat# 293-VE), Asparaginase (Sigma-Aldrich, Cat# A3809), l-Histidinol (Sigma-Aldrich, Cat# H6647), FFA (Sigma-Aldrich, Cat# F9905), α-glycyrrhetinic acid (Sigma-Aldrich, Cat# G10105), A438079 (Sigma-Aldrich, Cat# A9736), Oligomycin A (Sigma-Aldrich, Cat# 75351), CCCP (Sigma-Aldrich, Cat# C2759), antimycin A (Sigma-Aldrich, Cat# A8674), rotenone (Sigma-Aldrich, Cat# R8875), formate (Sigma-Aldrich, Cat# 06473), folate (Enzo Life Sciences, Cat# ALX-460-006). The following antibodies were used: pS6 (Cell Signaling, Cat# #2215, dilution 1:1000), S6 (Cell Signaling, Cat# 2317, dilution 1:1000), MTHFD2 (Proteintech, Cat# 12270-1-AP, WB dilution 1:1000, IF dilution 1:200), SHMT2 (Bethyl, Cat# A305-351A, dilution 1:2000), β-actin (Cell Signaling, Cat# 4970S, dilution 1:1000), Pecam-1 (BD Biosciences, Cat# 553370, dilution 1:200), FLAG (Sigma-Aldrich, Cat# F3165, dilution 1:1500). Primers for qRT-PCR are listed in Supplementary Table 9.