Anti flag m2

pEGFP-FORCP constructs were generated by cloning the ORF of FORCP-WT or mutant ATG of ORF into pEGFP-N1 vector with Age1/Not1 sites. For pLVX-FORCP 3xFLAG constructs, full length of FORCP-WT or FORCP-mut containing 3xFLAG in its C-terminus was subcloned into lentivirus vector pLVX-PURO with EcoR1/Xba1 sites. The BRI3BP-FLAG-Myc construct was purchased from OriGene technology, USA.
For transfection and immunostaining, 293 T cells were seeded at 300,000 cells per well in a six-well plate. After 24 hr, the cells were transfected using Lipofectamine 2000 (life technology Invitrogen) according the manufacturer’s instruction. Forty-eight hours after transfection, cells were reseeded onto chamber slides (Thermo Fisher Scientific, USA) and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Fixed cells were permeabilized by 0.5% Triton X-100 for 10 min at RT and stained with primary antibodies anti-FLAG M2 (Cell Signaling, rabbit), PDI (Sigma, mouse) or anti-FORCP custom antibody (Abgent) overnight at 4°C. After washing the cells three times with PBS, secondary antibody was added and incubated at RT for 1 hr. DNA was stained with DAPI (blue). Images were taken using a confocal microscope (Zeiss LSM 880 NLO Airyscan).

HEK293T, B16F10 (High Metastatic Mouse Melanoma), and LLC1 (Lewis Lung Carcinoma) cell lines were from ATCC between 2002 and 2003. Functional-grade anti-mouse CD3ϵ (145-2C11) and CD28 (37.51) were from BD Bioscience. Antibodies for phospho-Lck (Tyr505), phospho-SLP76 (Ser376), phospho-Zap70 (Tyr329)/Syk (Tyr352), phospho-PLCγ1 (Tyr783), phospho-LAT (Tyr220), Lck, LAT (E3U6), CD3ϵ (CD3-12) and anti-Flag (M2) were from Cell Signaling Technology. The rabbit anti-CCDC134 (ab106442) antibody was from Abcam Biotechnology (Cambridge, MA). Biotin anti-mouse CD3ϵ and biotin anti-mouse CD71 (transferrin receptor, TfR) were from Biolegend. Anti-His, and anti-HA antibodies were from Proteintech, and anti-β-actin (C-4) was from ZSGB-BIO.
Fluorescently labeled anti-mouse CD4 (L3T4), CD8 (53-6.7), CD3 (145-2C11), TCRβ (H57-597), CD44 (IM7), CD62L (MEL-14), CD45 (30-F11), CD127 (A7R34), KLRG1 (2F1), PD1 (29F.1A12), Tim3 (B8.2C12) and IFN-γ (XMG1.2) antibodies were purchased from Biolegend. Recombinant mouse IL-2 was from Peprotech. The reagents for mouse IL-2, TNF-α, and IFN-γ ELISAs were from eBioscience.

Transfected COS-7 cells were washed three times with ice cold PBS and scraped in buffer K (1 mM Tris-HCl, 1 mM EGTA and 1 mM MgCl₂, pH 7.6) containing protease inhibitor cocktail (Sigma, # P2714). Cells were mechanically lysed by passing them 10 times with 30_1/2-gauge needle and small fractions were used to measure the total protein using a Coomassie protein assay (Thermo Fisher Scientific, #1856209). Cell lysate was incubated with anti-FLAG M2 antibody for 1 h and immunoprecipitated (IP) using (protein A/G) agarose beads (Santa-Cruz Biotechnology, # SC2003). The supernatants were used to detect actin via western blotting and served as loading controls. Both the supernatant and immunoprecipitated fractions were subjected to electrophoresis on an 8% SDS-PAGE gel. The weight separated proteins were transferred to nitrocellulose membranes and probed with either anti-FLAG M2 (1:1000) or anti-PDI (Cell Signaling Technology, #3501 (1:1000)), anti-actin (Thermo Fisher Scientific, #PA1-183, (1:3000)) prepared in 2% BSA in TBS. The blots were washed and probed with HRP-conjugated corresponding secondary antibodies (goat anti-rabbit, Cell Signaling Technology, #7074, 1:5000 or goat anti-mouse, Thermo Fisher Scientific, #62–6520, 1:5000). The blots were developed in ChemiDoc^TM-Touch Imaging system from Bio Rad.

Transfected 293T cells were lysed with Cell Signaling lysis buffer (Cat#40–040, Millipore, Bellerica, USA). Protein concentration was determined via BCA analysis kit (ThermoScientific, Waltham, USA). Approximately 50ug of protein was loaded into a 12% SDS/Polyacrylimide gel. Proteins were transferred to nitrocellulose blotting paper via the dry HEP-OWL1 system (ThermoScientific, Waltham, MA). Nitrocellulose blots were incubated with Anti-Flag M2 (Cat#2368S, Cell Signaling, Danvers, MA) and anti-Actin (Cat#4967, Cell Signalling, Danvers, USA) antibodies. Bands were visualized via the Odyssey luminescence scanner (Li-Cor, Lincoln, USA) according to manufacturer’s instructions.