Anti ctla 4

SW480 cells were incubated and lysed on ice for 40 min. Protein was extracted and the concentrations were measured using the Bradford assay. Lysates were then separated by 12% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Sigma-Aldrich, USA) and incubated with the indicated antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad, Hercules, California, USA). An anti-GAPDH antibody was used as a control. Anti-INHBA, anti-PD-L1, anti-CTLA-4 and anti-GAPDH were purchased from Cell Signaling Technology, Inc (Beverly, Massachusetts, USA).

For western blot analysis of VPAC1, VPAC2, PD1 and CTLA4 expression, anti-VPAC1 (1:500), anti-VPAC2 (1:500) monoclonal antibodies from Sigma Aldrich (St. Louis, MO) and anti-PD1 (1:1000) and anti-CTLA-4 (1:500) monoclonal antibodies from Cell Signaling Technology (Danvers, MA) were used. For IF, anti-VIP monoclonal antibody at 1:50 from OriGene (Clone OT15B5) and anti-CK18 monoclonal antibody at 1:400 from Abcam (Clone EP1580Y) was used. Details of fluorochrome conjugated antibodies for flow cytometric analysis are provided in Supplementary Table 2. Fixable Aqua live/dead stain from Thermo Fisher Scientific (Waltham, MA) was used to detect and gate for live cells in all samples analyzed via flow cytometric analysis. To identify tumor specific T cells, APC conjugated MHC Tetramer H-2kb MuLV p15E from MBL International Corporation (Woburn, MA) was used. For intracellular cytokine expression staining, splenocytes or T cells were incubated with leukocyte activation cocktail (BD) for 5 h, then stained with antibodies listed in Supplementary Table 2. FACS files were acquired with a FACS Aria cytometer (Beckon Dickinson, San Jose, CA) or an Aurora cytometer (Cytek Biosciences, Inc, Fremont, CA) and analyzed using FlowJo software (Tree Star, Inc).