Fig 1: Characterization of CSF1R variants in vitro. (A) Immunoblotting of CSF1R‐FLAG in transfected HeLa cells as indicated. fl, Full‐length form; cl, cleaved form. (B) Quantification of A. n = 3 per group. (C) Quantitative analysis of mRNA expression after indicated transfection. n = 4 per group. WT, wild‐type. (D) Immunofluorescence staining of CSF1R‐FLAG overexpressing cells and counterstaining with actin‐tracker Rhodamine. DAPI labels the nuclei. Augmented area in the dashed boxes was shown. Scale bar, 20 μm. (E) Immunofluorescence staining of CSF1R‐FLAG and endoplasmic reticulum marker Calnexin in transfected HeLa cells. DAPI labels the nuclei. Augmented area in the dashed boxes was shown. Scale bar, 20 μm. (F) Immunoblotting detection of the level of tyrosine phosphorylation of CSF1R in transfected HEK293T cells after CSF1 treatment as indicated. IP, immunoprecipitation; sh, short exposure; lo, long exposure. (G) CCK8 assay suggests suppressed cell viability induced by the mutants. n = 8 per group. (H) Immunoblotting of pERK1/2 and total ERK1/2 in transfected HeLa cells. (I) Quantification of H. n = 3 per group. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (J) ERK1/2 activation after CSF1 treatment in CSF1R‐transfected HEK293T cells. (K) Diverse degree of ERK1/2 activation after acute CSF1 treatment in HEK293T cells transfected with indicated mutants. lo, long exposure; sh, short exposure. (L) Quantification of K. Fold changes were normalized to untreated CSF1R (WT)‐transfected group. n = 3 per group. *p < 0.05.