Fig 2: BLNK affects anchorage-independent growth of NCIH2122 and HGF-induced chemotaxis of NCIH1568(A) Validation of NCIH2122 BLNK CRISPR knockout cell lines via western blotting is shown. Total Met (a-Met), BLNK (a-BLNK), phosphorylation of ERK (a-pERK) and total ERK (a-ERK) are represented. GAPDH (a-GAPDH) was used as loading control.(B) (Left panel) Soft agar colony formation assay of NCIH2122 BLNK CRISPR knockout clonal cell lines, with parental NCIH2122 and NCIH2122 AAVS1 targeting control shown. Wells with no cells were used as the negative control. Results are representative of n = 3 technical replicates. (Right panel) Number of colonies counted using ImageJ. Significance was assessed using unpaired two-tailed Welch’s t test. Results are shown as mean ± SD, with n = 3 technical replicates. *p<0.05; **p<0.01.(C) (Right panel) Transwell migration assay investigating the migration of NCIH1568 (NSCLC) cells toward either no HGF or 50 ng/mL HGF in response to siRNA-mediated knockdown of BLNK is shown. (Middle panel) Quantification of Crystal violet staining is shown. Significance was assessed using unpaired two-tailed Welch’s t test. Results are shown as mean ± SD, with n = 2 technical replicates *p<0.05; (Right panel) Knockdown of BLNK was verified using western blotting. Met (a-Met) and BLNK (a-BLNK) are shown. GAPDH (a-GAPDH) was used as loading control.

Fig 3: BLNK is a regulator of Met signaling(A) Time course signaling assay in response to HGF in FLAG-BLNK- and empty vector-transfected HEK293 cells is shown. Total Met (a-Met), BLNK (a-FLAG), phosphorylation of ERK (a-pERK) and total ERK (a-ERK) are represented. GAPDH (a-GAPDH) was used as loading control.(B) Time course signaling assay in response to HGF in NCIH2122 cells with siRNA-mediated knockdown of BLNK is shown. Total Met (a-Met), BLNK (a-BLNK), phosphorylation of ERK (a-pERK), total ERK (a-ERK), phosphorylation of AKT (a-pAKT) and total AKT (a-AKT) are represented. GAPDH (a-GAPDH) was used as loading control.(C) Time course signaling assay in response to HGF in NCIH1568 cells with siRNA-mediated knockdown of BLNK is shown. Total Met (a-Met), BLNK (a-BLNK), phosphorylation of ERK (a-pERK), total ERK (a-ERK), phosphorylation of AKT (a-pAKT) and total AKT (a-AKT) are represented. GAPDH (a-GAPDH) was used as loading control.(D)and(E) Phosphorylation of Met in response to HGF post siRNA-mediated knockdown of BLNK is assessed using immunoprecipitation of Met in NCIH2122 (D) and NCIH1568 (E). Total Met (a-Met), phosphorylated Met (a-pMet Y1234/Y1235) and BLNK (a-BLNK) are shown. GAPDH (a-GAPDH) was used as loading control.

Fig 4: BLNK is a regulator of HGF-independent and-dependent subcellular localization of Met(A) Surface Biotinylation assay detecting surface Met levels of NCIH2122 in response to siRNA-mediated knockdown of BLNK is shown. Whole cell lysate (WCL) and surface Met (a-Met) and BLNK (a-BLNK) are represented. Tubulin (a-Tubulin) was used as loading control.(B) Surface Biotinylation assay detecting surface Met levels of NCIH1568 in response to siRNA-mediated knockdown of BLNK is shown. Whole cell lysate (WCL) and surface Met (a-Met) and BLNK (a-BLNK) are represented. Tubulin (a-Tubulin) was used as loading control.(C) (Left panel) Immunofluorescence to assess localization of Met at 0- and 30 min of HGF stimulation in response to siRNA-mediated knockdown of BLNK in NCIH2122 is shown. Scale bar = 10µM (Middle panel) Average distance of Met puncta to the plasma membrane is plotted. Individual dots represent one cell. Significance was assessed with one way ANOVA. Data is represented as mean ± SD for n = 45 cells; ns = not significant, ****p<0.0001. (Right panel) Knockdown of BLNK was verified using western blotting. Met (a-Met) and BLNK (a-BLNK) are shown. Actin (a-Actin) was used as loading control.(D) (Left panel) Immunofluorescence to assess localization of Met at 0-, 15- and 30 min of HGF stimulation in response to siRNA-mediated knockdown of BLNK in NICH1568 is shown. Scale bar = 10µM (Middle panel) Relative distance of Met puncta to the plasma membrane is plotted. Individual dots represent one cell. Significance was assessed with one-way ANOVA. Data is represented as mean ± SD for n = 45 cells; ns = not significant, ****p<0.0001. (Right panel) Knockdown of BLNK was verified using western blotting. Met (a-Met) and BLNK (a-BLNK) are shown. Actin (a-Actin) was used as loading control.

Fig 5: Met-GRB2 interaction is increased in the presence of BLNK, and BLNK-GRB2 interaction is increased in the presence of active Met(A) (Left panel) Schematic of modified MaMTH assay is shown. Created with Biorender.com. (Right panel) MaMTH assay examining the interaction between Met and GRB2 or PEX7 (negative control) in the presence of GFP-tagged BLNK or GFP-tagged LCP2 is shown. Significance was assessed using unpaired two-tailed Welch’s t test. Results are shown as mean ± SD, with n = 4 technical replicates. ***p<0.001. Protein expression of all constructs was detected using western blotting. Met bait (a-V5), GRB2/NCK2 preys (a-FLAG) and BLNK/LCP2 (a-GFP) are shown. GAPDH (a-GAPDH) was used as a loading control.(B) Co-immunoprecipitation investigating interaction between GRB2 and BLNK in the presence of Met under different conditions is shown. Phosphorylated Met was detected using a-pY antibody. Tubulin (a-Tubulin) was used as a loading control. IP-Immunoprecipitated fraction; WCL – Whole cell lysate.(C) SIMPL-ELISA assay examining interaction between GRB2 bait and various BLNK mutants or PEX7 (negative control) preys is shown. Mock - no transfection, empty - empty vector transfection. The spliced signal is expressed as relative luciferase units and is normalized to bait and prey expression. Grubbs test was performed to remove any outliers. Significance was assessed using unpaired two-tailed Student’s t test. Results are shown as mean ± SD, with n= minimum of 2 technical replicates after Grubbs test. *p<0.05; **p<0.01; ***p<0.001.(D) Time course signaling assay in response to HGF, in the presence of BLNK wildtype, BLNK P204A212AY72F or empty vector in HEK293 cells is shown. Total Met (a-Met), BLNK (a-FLAG), phosphorylation of ERK (a-pERK) and total ERK (a-ERK) are represented. GAPDH (a-GAPDH) was used as loading control.