phospho alk  (Cell Signaling Technology Inc)

Journal: Signal Transduction and Targeted Therapy

Article Title: RNase1-driven ALK-activation is an oncogenic driver and therapeutic target in non-small cell lung cancer

doi: 10.1038/s41392-025-02206-x

Figure Lengend Snippet: RNase1 binds to and activates ALK as its ligand in lung cancer cells. a Each of the 13 recombinantly purified 6´ N-terminal His-tagged RNases (10 µg) was incubated with H1299 lysate followed by Ni-His beads pull down and Western blot analysis with the indicated antibodies. b Each of the 13 RNases was added into the H1299 culture medium at a final concentration of 1 mg/ml. After 30 min incubation, cells were lysed and subjected to Western blotting with the indicated antibodies. A phospho-ALK antibody was used to detect ALK Y1604 phosphorylation. c Western blot analysis of ALK phosphorylation sites in HEK293 cells. Flag-tagged wild type or Tyr mutant ALK plasmids were transfected into HEK293 cells and then IP with Flag-beads. A pan-phosphorylated Tyr antibody (4G10) was used to detect ALK phosphorylation. d Plasmids expressing Myc-tagged ALK and C-terminal Flag-tagged RNase1 (R1) were transfected into HeLa cells. Cell lysates were harvested and subjected to co-immunoprecipitation (co-IP) assay. RNase1 was detected using the Flag antibody. RNase5 (R5) was used as a negative control. e In vitro binding affinity assay of ALK and RNase1. Kd, dissociation constant. BSA was used as a negative control. f Immunofluorescence microscopy of H1299 cells with or without RNase1 expression. Yellow dots and white arrows both indicate co-localization of ALK and RNase1. Scale bar, 20 mm. g H1299 cells with or without RNase1 expression were subjected to Duo Link assay. Red dots indicate binding between ALK and RNase1. Scale bar, 20 mm. h Time course analysis of ALK activation in H1299 cells by RNase1 (1 mg/ml). ALK phos-Y1604 was used as an indicator of ALK activation. i Co-IP of ALK and RNAse1 from HEK293T cells transfected with wild-type (WT) or catalytic-deficient (CD) RNase1-expressing plasmid followed by Western blotting with the indicated antibodies

Article Snippet: Antibodies against RNase1 (Sigma HPA001140), ALK (Cell Signaling 3633), phosphorylated ALK (Tyr1604; Cell Signaling 3341S), phosphorylated ERK1/2 (Thr202/Tyr204; Cell Signaling 9101), ERK1/2 (Cell Signaling 9102), STAT3 (Cell Signaling 9132), phosphorylated STAT 3 (Cell Signaling 9145S), PD-L1 (Cell Signaling 13684 s), and tubulin (Sigma B-5-1-2) were used for immunoblotting, immunohistochemistry, immunofluorescence, and Duolink assays.

Techniques: Purification, Incubation, Western Blot, Concentration Assay, Phospho-proteomics, Mutagenesis, Transfection, Expressing, Co-Immunoprecipitation Assay, Negative Control, In Vitro, Binding Assay, Immunofluorescence, Microscopy, Activation Assay, Plasmid Preparation