Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: IKKε directly phosphorylates kindlin-2 at S159. (A) Slot blot analysis of the anti-phospho-kindlin-2-S159 antibody. The antibody was probed against an un-modified kindlin-2 peptide and phosphorylated peptides. (B) HCT116 cells were transfected with the indicated plasmids and whole cell extracts were immunoprecipitated (IP) with FLAG-conjugated M2 beads and then analyzed by immunoblotting (IB). (C)HCT116 cells were treated with the inhibitor IKK-3 at the indicated concentration for 24h and analyzed by immunoblotting. (D and E) HCT116 cells were transfected with the indicated siRNAs targeting IKKα, IKKβ, IKKε or TBK1. Cell lysates were analyzed by immunoblotting. (F) HCT116 cells were transfected with FLAG-tagged kindlin-2 and GFP-tagged IKKε for 48 h. Total cell lysates were immunoprecipitated using FLAG-conjugated M2 beads and analyzed by immunoblotting. (G) IKKε was immunoprecipitated using anti-IKKε antibody or the control antibody IgG from lysates of HCT116 cells. (H) HCT116 cells were transfected with the indicated plasmids and the whole cell extracts were prepared and analyzed by Co-IP assay flowed by immunoblotting with the indicated antibodies. (I) GST-tagged wild-type kindlin-2 or kindlin-2 (S159A) were incubated without IKKε (-), or with purified wild-type IKKε (WT) or mutant IKKε (K38A) in kinase buffer and resolved by SDS-PAGE, followed by immunoblotting analysis with the indicated antibodies.

Article Snippet: The following antibodies were used in this study: anti-kindlin-2-S159 (CTM-152, PTM Biolabs) ; anti-kindlin-2 (K3269) and anti-FLAG (F3165) (Sigma-Aldrich); anti-IKKα (2682), anti-IKKβ (2678), anti-IKKε (2905), anti-TBK1 (3504) (Cell Signaling Technology); anti-GAPDH (sc-3223), anti-FISH (sc-376211) and anti-cortactin (sc-55579) (Santa Cruz); anti-IKKε (ab7891) (Abcam); anti-SH3PXD2A (18976-1-AP) (Proteintech); anti-MIG2/Kindlin-2 (NBP1-47745) (NOVUS).

Techniques: Dot Blot, Modification, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Co-Immunoprecipitation Assay, Incubation, Purification, Mutagenesis, SDS Page

Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: IKKε-induced invadopodia formation depends on kindlin-2 S159 phosphorylation. (A) HCT116 cells were transfected with GFP-tagged IKKε and stained with antibodies against kindlin-2, Tks5 and F-actin (phalloidin). Confocal images were acquired to visualize the localization of IKKε, kindlin-2, Tks5 and F-actin. White arrowhead point to the co-localization of IKKε, kindlin-2, Tks5 and F-actin. Scale bar, 10 μm. (B) Co-localization of Tks5 and F-actin in HCT116 cells with kindlin-2 knockout and rescue with wild-type IKKε, wild-type kindlin-2, phosphor-null mutant kindlin-2 (S159A) and phosphor-mimetic mutant (S159D) kindlin-2 (left). Inset shows magnified image of invadopodia in the box. Quantification of cells with invadopodia (right). (C) Gelatin degradation in HCT116 cells with kindlin-2 knockout and rescue with wild-type IKKε, wild-type kindlin-2, phosphor-null mutant kindlin-2 (S159A) and phosphor-mimetic mutant (S159D) kindlin-2 (left). The area of degraded gelatin per cell (right). All data represent the means ± S.D. (N>400 cells/sample, * P <0.05, ** P < 0.01, and *** P < 0.001).

Article Snippet: The following antibodies were used in this study: anti-kindlin-2-S159 (CTM-152, PTM Biolabs) ; anti-kindlin-2 (K3269) and anti-FLAG (F3165) (Sigma-Aldrich); anti-IKKα (2682), anti-IKKβ (2678), anti-IKKε (2905), anti-TBK1 (3504) (Cell Signaling Technology); anti-GAPDH (sc-3223), anti-FISH (sc-376211) and anti-cortactin (sc-55579) (Santa Cruz); anti-IKKε (ab7891) (Abcam); anti-SH3PXD2A (18976-1-AP) (Proteintech); anti-MIG2/Kindlin-2 (NBP1-47745) (NOVUS).

Techniques: Transfection, Staining, Knock-Out, Mutagenesis

Journal: Theranostics

Article Title: IKKε phosphorylates kindlin-2 to induce invadopodia formation and promote colorectal cancer metastasis

doi: 10.7150/thno.40397

Figure Lengend Snippet: Phosphorylated kindlin-2 (S159) levels positively correlate with IKKε expression in colorectal cancer tissues. (A) Immunohistochemical staining for phosphor-kindlin-2-S159 in human colorectal cancer tissues and paired adjacent normal tissues. Scale bar, 200 μm. (B) Phosphor-kindlin-2-S159 protein levels in 161 paired human colorectal cancer tissues and adjacent normal tissues as measured by immunohistochemical analysis. The data represent the means ± S.D. (*** P < 0.001). (C) Representative images of paraffin tissue sections stained with IKKε and phosphorylated kindlin-2 (S159) antibody. Scale bar, 200 μm. (D) Correlation analyses of IKKε and phosphorylated kindin-2 (S159) protein levels in human colorectal cancer specimens.

Article Snippet: The following antibodies were used in this study: anti-kindlin-2-S159 (CTM-152, PTM Biolabs) ; anti-kindlin-2 (K3269) and anti-FLAG (F3165) (Sigma-Aldrich); anti-IKKα (2682), anti-IKKβ (2678), anti-IKKε (2905), anti-TBK1 (3504) (Cell Signaling Technology); anti-GAPDH (sc-3223), anti-FISH (sc-376211) and anti-cortactin (sc-55579) (Santa Cruz); anti-IKKε (ab7891) (Abcam); anti-SH3PXD2A (18976-1-AP) (Proteintech); anti-MIG2/Kindlin-2 (NBP1-47745) (NOVUS).

Techniques: Expressing, Immunohistochemical staining, Staining