anxa1  (Proteintech)

Journal: The FASEB Journal

Article Title: A Novel Dysferlin‐Binding Kinase CK2α Promotes Plasma Membrane Repair in Dysferlinopathy

doi: 10.1096/fj.202500773RRR

Figure Lengend Snippet: Phosphorylation of annexin A1 is decreased in human myocytes with dysferlin mutations. (A) immunoblots of annexin A1, Dysferlin and Vinculin in human myoblasts and myotubes. Line 107 and 379 have dysferlin mutations. (B) The bands of annexinA1 were normalized to those of vinculin and semiquantified. n = 3, Tukey–Kramer test. n.s., Not significant. (C) PhosTag of annexin A1 in human myoblasts. The black line represents the phosphorylation band.

Article Snippet: The antibodies used for immunoblotting and immunoprecipitation were as follows: HA tag (51064–2‐AP; Proteintech, Rosemont, IL), Halo tag (G9281; Promega), dysferlin (NCL‐Hamlet, Leica, Wetzlar, Germany), CK2α/α′ (MCA3031Z; Bio‐Rad), CK2β (ab76025; Abcam, Cambridge, UK), annexin A1 (21990–1‐AP, Proteintech) (#610067, BD Bioscience, Franklinlakes, NJ), pAkt (S127) (ab133458; Abcam), Akt (#9272; CST, Danvers, MA), GFP (M048‐3; MBL, Tokyo, Japan), GAPDH (#2118; CST), Vinculin (#13901; CST), and HRP‐conjugated secondary antibodies (mouse: #7076; CST, rabbit: #7074; CST).

Techniques: Phospho-proteomics, Western Blot

Journal: The FASEB Journal

Article Title: A Novel Dysferlin‐Binding Kinase CK2α Promotes Plasma Membrane Repair in Dysferlinopathy

doi: 10.1096/fj.202500773RRR

Figure Lengend Snippet: Identified annexin A1 as a candidate substrate for CK2α. (A) Peptides were recovered from wild‐type and CK2α KO C2C12 myoblast cells, followed by enrichment of phosphorylated peptides with titanium beads and subsequent mass spectrometry analysis. (B) The MA plot was constructed with log2 ([CK2α KO]/[WT]) on the horizontal axis and the mean amount of detected peptides on the vertical axis. Phosphorylated peptides showing a significant decrease with CK2α KO were identified as those decreasing below −1.0 on the horizontal axis (indicated by the red line). (C) A list of phosphorylated peptides detected in this study, which are reported to be involved in cell membrane repair. (D) With a Qual Browser, the detection intensity of the phosphorylated peptides of annexin A1 was compared between the WT and CK2α KO strains. (E) PhosTag was utilized to assess the phosphorylation of annexin A1 in WT and CK2α KO C2C12 myoblasts. The black line represents the phosphorylation band observed in the WT.

Article Snippet: The antibodies used for immunoblotting and immunoprecipitation were as follows: HA tag (51064–2‐AP; Proteintech, Rosemont, IL), Halo tag (G9281; Promega), dysferlin (NCL‐Hamlet, Leica, Wetzlar, Germany), CK2α/α′ (MCA3031Z; Bio‐Rad), CK2β (ab76025; Abcam, Cambridge, UK), annexin A1 (21990–1‐AP, Proteintech) (#610067, BD Bioscience, Franklinlakes, NJ), pAkt (S127) (ab133458; Abcam), Akt (#9272; CST, Danvers, MA), GFP (M048‐3; MBL, Tokyo, Japan), GAPDH (#2118; CST), Vinculin (#13901; CST), and HRP‐conjugated secondary antibodies (mouse: #7076; CST, rabbit: #7074; CST).

Techniques: Mass Spectrometry, Construct, Membrane, Phospho-proteomics

Journal: The FASEB Journal

Article Title: A Novel Dysferlin‐Binding Kinase CK2α Promotes Plasma Membrane Repair in Dysferlinopathy

doi: 10.1096/fj.202500773RRR

Figure Lengend Snippet: Schematic diagram of plasma membrane repair mediated by dysferlin, as elucidated in this study. (A) At steady state, dysferlin spans the membrane at the C‐terminus, and CK2 binds to dysferlin at region II. (B) When the cell membrane is injured, extracellular calcium ions influx, leading to the accumulation of dysferlin at the injury site, where it is believed to bind to the cytoplasmic side of the membrane via its C2 domain. In the presence of dysferlin, CK2 also efficiently distributes to the site of membrane injury, maintaining its kinase activity by interacting with dysferlin. Phosphorylated annexin A1 recruits intracellular vesicles to the injury site. (C) The mobilized intracellular vesicles are hypothesized to serve as a source of phospholipids, which act as material for the patch that seals the damaged site in cellular membrane repair.

Article Snippet: The antibodies used for immunoblotting and immunoprecipitation were as follows: HA tag (51064–2‐AP; Proteintech, Rosemont, IL), Halo tag (G9281; Promega), dysferlin (NCL‐Hamlet, Leica, Wetzlar, Germany), CK2α/α′ (MCA3031Z; Bio‐Rad), CK2β (ab76025; Abcam, Cambridge, UK), annexin A1 (21990–1‐AP, Proteintech) (#610067, BD Bioscience, Franklinlakes, NJ), pAkt (S127) (ab133458; Abcam), Akt (#9272; CST, Danvers, MA), GFP (M048‐3; MBL, Tokyo, Japan), GAPDH (#2118; CST), Vinculin (#13901; CST), and HRP‐conjugated secondary antibodies (mouse: #7076; CST, rabbit: #7074; CST).

Techniques: Clinical Proteomics, Membrane, Activity Assay

Journal: PLOS Pathogens

Article Title: Genome-wide CRISPR screening identifies Annexin A1 as a facilitator of porcine astrovirus entry

doi: 10.1371/journal.ppat.1013943

Figure Lengend Snippet: (A) Western blot validating ANXA1 overexpression in PK15 and IPEC-J2 cells using mouse anti-ANXA1 antibody. (B) RT-qPCR analysis of PAstV infection (MOI = 0.01) at 24 h in ANXA1-overexpressing PK15 and IPEC-J2 cells. (C) Schematic design of CRISPR-resistant ANXA1 (pANXA1). (D) Western blot confirming ANXA1 restoration in PK15-ANXA1KO after PAstV infection. (E) RT-qPCR analysis of PAstV infection (MOI = 0.01) at 24 h in ANXA1-rescued PK15 cells. (F) RT-qPCR analysis of PAstV infection (MOI = 0.01) at 24 h in ANXA1 polyclonal KO IPEC-J2 cells. (G) Immunofluorescence detection of virus replication in PK15-WT and PK15-ANXA1 KO cells. (H) RT-qPCR analysis of virus replication (MOI = 0.1) in PK15-ANXA1 KO cells at 24 h post-infection. (I, J) Western blot (I) and RT-qPCR (J) analysis of ANXA1 expression in PK15 cells infected with PAstV (MOI = 1). Data represent mean ± SD (n = 3). Statistical significance by unpaired two-tailed Student’s t-test and Two-way ANOVA. (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001).

Article Snippet: Primary antibodies used included anti-ANXA1 (21990–1-AP), anti-RIG-I (67556–1-Ig), anti-IRF3 (11312–1-AP), anti-phospho-IRF3 (29528–1-AP), anti-caspase 3/P17/P19 (82202–1-RR), anti-Flag (66008–4-Ig), anti-beta-tubulin (10094–1-AP), and anti-HA-HRP (HRP-81290) were purchased from Proteintech, China.

Techniques: Western Blot, Over Expression, Quantitative RT-PCR, Infection, CRISPR, Immunofluorescence, Virus, Expressing, Two Tailed Test