Foxp1

Endothelial cells were lysed in modified Radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris pH 8, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium desoxycholate, 1% Triton X-100, 150 mM NaCl, 1× protease inhibitor cocktail). Proteins were separated by SDS–polyacrylamide gel electrophoresis gradient (4–20%) gel and transferred onto nitrocellulose membranes and incubated overnight at 4°C with primary antibodies. The following primary antibodies were used in this study: PLVAP (DSHB, Cat#MECA-32); PTGS1 (Cell Signaling Technologies, Cat#9896S); TXNIP (Cell Signaling, Cat#71632); FOXP1 (Cell Signaling Technologies Cat#4402S); TFRC (DSHB, Cat#G1/221/12); ZFP36L2 (Cell Signaling, Cat#2119); ACTN1 (Sigma, Cat#A5044); GAPDH (Millipore Sigma, Cat#MAB374); VCL (Millipore Sigma, Cat#V-9131). Secondary antibodies included: Amersham ECL Rabbit IgG HRP-Linked Whole Antibody (Cat#NA934) and Amersham ECL Mouse IgG, HRP-Linked Whole Antibody (Cat#NA931) both from Cytiva. Immuno-complexes were detected by enhanced chemiluminescence with SuperSignal West Pico PLUS (Thermo Fisher Scientific #PI34580) and Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific #PI34096) using ChemiDoc Touch Imaging System (Bio-Rad Laboratories). Quantification of bands by densitometry analysis was performed using ImageLab Software (Bio-Rad Laboratories).

Whole cell lysates were extracted using RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 250 mM NaCl, 0.1% SDS, 0.5% NP-40, 2 mM DTT, 1 mg/mL protease inhibitors). Protein concentration was quantified using a BCA protein assay kit (Pierce, Grand Island, NY). Afterward, 50 μg of protein was resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted onto a PVDF membrane (Millipore, Billerica, MA, USA). Immunodetection using antibodies against FOXP1 (Cell Signaling, Danvers, MA), NF-κB p65 (Abcam, MA, USA), or β-actin (Millipore) was done for 1 h at room temperature or overnight at 4°C. Antibody-antigen complexes were detected using the enhanced chemiluminescence (ECL) system (Amersham Bioscience, Piscataway, NJ, USA). β-actin served as a loading control.

Whole-cell extracts from mESCs cultured in feeder-free 2i media were prepared in a modified RIPA lysis buffer: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1x SigmaFast protease inhibitor cocktail. Spheroids were lysed in RIPA buffer and protease inhibitors. Protein concentration was determined with a BCA kit (Thermo Fisher Scientific) and normalized to 1.0–2.0 μg/μl. 20–25 μl of protein was mixed with sample buffer and 50–100 mM DTT. Protein was transferred to a PVDF membrane, blocked with either 1% BSA or Intercept (TBS) Blocking Solution (LI-COR), and blotted with primary antibodies at 4 °C overnight. Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204). Western blots were imaged on an Odyssey DLx Imager system.