phospho src tyr416  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Src-mediated PHB2 phosphorylation disrupts mitochondrial cristae through cardiolipin dissociation in hepatocellular carcinoma

doi: 10.1016/j.redox.2026.104073

Figure Lengend Snippet: Src kinase specifically phosphorylates PHB2 at Y34 and Y77 in hepatocellular carcinoma cells. A,B Differential phosphorylation of PHB proteins in LO2 versus HepG2 cell lines. ( A ) PHB1 phosphorylation analysis. ( B ) PHB2 phosphorylation analysis. Upper panels: Immunoprecipitation (IP) with anti -PHB1 or anti -PHB2 antibodies followed by detection of phosphorylated proteins (phos-PHB1/2) and total PHB1/2. IgG serves as negative control. Lower panels: Input lysates showing total PHB1/2 and β-actin loading control. C Identification of kinases involved in PHB2 phosphorylation in HepG2 cells. HepG2 cells expressing PHB2-WTFlag were transfected with siRNA targeting INSR, SRC, or EGFR, or scramble control. Upper panel: IP with anti -PHB2 antibody followed by Western blot detection of phos-PHB2, total PHB2, and IgG control. Lower panel: Input lysates showing PHB2 and β-Actin (loading control). D Effect of SRC overexpression on PHB2 phosphorylation in HepG2 cells. HepG2 cells expressing PHB2-WT Flag were transduced with control (SRC-NC) or SRC-overexpressing lentivirus. IP-Western blot analysis as described in (B). E Effect of SRC knockdown on PHB2 phosphorylation in HepG2 cells. HepG2 cells expressing PHB2-WT Flag were transfected with scramble control or SRC-specific shRNA. IP-Western blot analysis as described in (B). F Identification of SRC phosphorylation sites on PHB2 in HepG2 cells. HepG2 cells were transfected with various Flag-tagged PHB2 mutants (Y34F, S39A, Y77F, Y34F/S39A, Y34F/Y77F, S39A/Y77F) with or without SRC overexpression. IP-Western blot analysis shows phosphorylation levels of different PHB2 mutants. All immunoprecipitation and Western blot experiments were performed in at least three independent experiments with similar results. Representative blots are shown. G The KM survival curve of the Src gene in TCGA data, where different groups are tested using the log-rank test. HR (High exp) represents the hazard ratio of the high expression group relative to the low expression group.

Article Snippet: The following antibodies were used: anti -PHB2 (1:1000, Cell Signaling Technology #14085), anti -PHB1 (1:1000, Abcam ab75771), anti -phosphotyrosine (1:1000, Cell Signaling Technology #9411), anti -SRC (1:1000, Cell Signaling Technology #2109), anti -β-actin (1:5000, Sigma-Aldrich A5441), anti -HSP60 (1:1000, Cell Signaling Technology #12165), anti -OMA1 (1:1000, Santa Cruz Biotechnology sc-515788), anti -OPA1 (1:1000, BD Biosciences 612606), anti -VDAC (1:2000, Proteintech #66345‐1‐Ig), and anti -AFG3L2 (1:1000, Abcam ab139503).

Techniques: Phospho-proteomics, Immunoprecipitation, Negative Control, Control, Expressing, Transfection, Western Blot, Over Expression, Transduction, Knockdown, shRNA

Journal: Redox Biology

Article Title: Src-mediated PHB2 phosphorylation disrupts mitochondrial cristae through cardiolipin dissociation in hepatocellular carcinoma

doi: 10.1016/j.redox.2026.104073

Figure Lengend Snippet: Src-mediated phosphorylation disrupts PHB1/2 complex stability and triggers PHB2 cytoplasmic mislocalization. A Electrostatic surface potential analysis of PHB2 showing the effect of phosphorylation. Left panel: wild-type PHB2 with Y34 and Y77 residues. Right panel: phosphorylated PHB2 (phos-Y34 and phos-Y77) showing altered electrostatic distribution. Blue indicates positive charge (+5), white is neutral (0), and red indicates negative charge (−5). The double-framed line represents the inner mitochondria membrane. B Effect of Src-mediated phosphorylation on PHB1/2 complex formation. Blue Native PAGE (BN-PAGE) analysis of PHB2-WT and phosphorylation-resistant mutant PHB2–Y34F/Y77F Flag in HepG2 cells with or without SRC overexpression. Protein load was verified by Coomassie blue (CBB) staining. Lower panel shows SDS-PAGE verification of SRC, PHB1, PHB2, and β-Actin expression. C Analysis of PHB1/2 complex formation in LO2 versus HepG2 cell lines. Upper panel: representative BN-PAGE showing PHB1/2-SC (supercomplex) and PHB1/2 complex formation detected with PHB2 and AFG3L2 antibodies. Lower panel: SDS-PAGE Western blot analysis of PHB2, AFG3L2, and β-Actin (loading control) expression levels. D Effect of OXPHOS inhibitors on PHB1/2 complex formation. Left panels: representative BN-PAGE analysis of PHB1/2-SC and PHB1/2 complexes in control and treated conditions (H 2 O 2 , Rotenone, Antimycin, Oligomycin (OLG), and combined Antimycin/Oligomycin (OA)). Protein load was verified by Coomassie blue (CBB) staining. Lower panel: representative Western blot showing protein expression levels of PHB2, AFG3L2, TIM23, and β-Actin (loading control). E Subcellular distribution of PHB2 under tumor microenvironment stresses. Western blot analysis of PHB2 in mitochondrial and cytoplasmic fractions of HepG2 cells treated with H 2 O 2 (oxidative stress), CoCl 2 (hypoxia mimetic), rotenone (Complex I inhibitor), or antimycin (Complex III inhibitor). F Dose-dependent PHB2 redistribution in response to increasing SRC expression levels. Western blot analysis showing PHB2 subcellular localization in mitochondrial and cytoplasmic fractions after SRC overexpression in HepG2 cells. HSP60 serves as mitochondrial marker and β-Actin as cytoplasmic marker (E, F). All Blue Native PAGE and Western blot experiments (B–F) were performed in at least three independent experiments. Representative images are shown.

Article Snippet: The following antibodies were used: anti -PHB2 (1:1000, Cell Signaling Technology #14085), anti -PHB1 (1:1000, Abcam ab75771), anti -phosphotyrosine (1:1000, Cell Signaling Technology #9411), anti -SRC (1:1000, Cell Signaling Technology #2109), anti -β-actin (1:5000, Sigma-Aldrich A5441), anti -HSP60 (1:1000, Cell Signaling Technology #12165), anti -OMA1 (1:1000, Santa Cruz Biotechnology sc-515788), anti -OPA1 (1:1000, BD Biosciences 612606), anti -VDAC (1:2000, Proteintech #66345‐1‐Ig), and anti -AFG3L2 (1:1000, Abcam ab139503).

Techniques: Phospho-proteomics, Membrane, Blue Native PAGE, Mutagenesis, Over Expression, Staining, SDS Page, Expressing, Western Blot, Control, Marker