Anti flag antibody

For pull-downs of overexpressed Arl3-Flag 2.5 × 10⁶ HEK293 cells were seeded in 15 cm² dishes 24 hr prior to transfection. Cells were transfected using Polyethylenimine (PEI) at a ratio 3:1 of PEI (µg) : total DNA (µg). Cells were induced to ciliate by withdrawing serum for 30 hr. ~2.5 × 10⁷ cells (1 × 15 cm² dish) were lysed in 1 ml lysis buffer for 30 min at 4°C. For pull-downs of endogenous Arl3 1 × 10⁸ cells (4 × 15 cm² dish) were used. Lysate was cleared by centrifugation and protein concentration normalized. Per sample 50 µg GST-PDE6δ was coupled to 50 µl glutathione agarose which was incubated with cleared lysates for 45 min at 4°C. Cleared lysate was removed and beads washed 2x with 500 µl buffer M. Samples were eluted with 1 × SDS-loading buffer. For the detection of affinity-precipitated endogenous Arl3 an anti-Arl3 antibody (Novus Biologicals) was used, and in case of Arl3-Flag an anti-Flag antibody (Thermo Scientific) was used. Expression of Arl13B-GFP was checked using an anti-GFP antibody (Santa Cruz Biotechnology) and antibody against S-peptide, which is located between Arl13B and GFP in pGLAP5. The level of Arl3·GTP was quantified using ImageJ. Experiments were repeated two or more times.

Flag-SH2B3 and Myc-JAK2/SHP2 plasmids were all designed and constructed from GeneChem Co., Ltd (Shanghai, China). Lung cancer cells transfected with Flag-SH2B3 and Myc-JAK2/SHP2 or naive lung cancer cells were lysed with lysis buffer supplemented with protease inhibitor. Protein concentration was quantified with Pierce BCA protein Assay (Thermo Fisher Scientific). An equal amount of proteins were incubated with anti-Flag antibody (Thermo Fisher Scientific, cat #MA1-91878, 1:500) or anti-SH2B3 antibody (Santa Cruz Biotechnology, cat #sc-393709, 1:100) for 1 h at 4 °C and then incubated with Protein-A Sepharose beads (Abcam, Cambridge, UK) overnight at 4 °C. The beads were then washed with lysis buffer followed by elution with sodium dodecyl sulfate (SDS) loading buffer. The elution was performed with standard western blot assay.

HEK293 cells transfected with wild‐type GPR21 and GPR21 mutants were harvested and incubated with phosphate buffer solution (PBS) plus 0.5% bovine serum albumin (BSA) for 15 min, followed by incubation with anti‐FLAG antibody (Thermo Fisher) at 4°C for 1 h. After washing with PBS three times, the cells were incubated with Alexa‐488‐conjugated secondary antibody (Beyotime) at 4°C for 1 h. After washing with PBS three times, the cells were subjected to flow cytometric analysis on an AccuriTM C6 Plus flow cytometer (BD). FL1 channel was employed to record fluorescent signal derived from Alexa‐488. The surface expression levels of GPR21 mutants were normalized to that of wild‐type GPR21 (100% level).