phos-fak (tyr 397) #8556 antibody  (Cell Signaling Technology Inc)

Journal: Life science alliance

Article Title: Tbx1 plays a critical role in focal adhesion dynamics through paxillin regulation.

doi: 10.26508/lsa.202403151

Figure Lengend Snippet: Figure 1. FA dynamics are altered in Tbx1-depleted cells. (A) Graphs showing the quantification of FA assembly and/or disassembly rate of Tbx1-depleted cells (Tbx1KD) or control cells (NT). (B) Photographs of frames obtained from time-lapse spinning confocal video microscopy movies. Indicated are some of the specific frames that were analysed to assess the number of forming or disassembling FA (arrows) in the lamellipodium of migrating cells, which were transfected with a construct encoding vinculin–GFP fusion protein to mark FAs. (C) Graph showing the number of unstable FA/cell normalized for total FA (FA remaining for less than 30 min). (D) Total Rac1 activity in collagen-stimulated cells was evaluated by the GST-RBD pull-down assay. The levels of total Rac1 or active Rac1 pulled down by GST-RBD analysed by immunoblotting with anti-Rac1 antibody. GAPDH was used as a loading control. (E) ECM-stimulated cells were analysed for the P-FAK (residues Y397 or Y925) and P-ERK1/2 levels by immunoblotting with specific antibody. GAPDH was used as a loading control. Graphs represent quantitative densitometric analysis from at least three experiments (right panels). N = 3 biological replicates. The scale bar represents 10 μm. *P < 0.05, **P < 0.01.

Article Snippet: Membranes were subsequently incubated for 1 h at RT in TBST buffer (125mM Tris–HCl [pH 8.0], 625 mM NaCl, 0.1% Tween-20) containing 5% BSA and further incubated at 4°C for 16 h with the following primary antibodies: phospho-Pxn (#2541; Cell Signaling), phospho-FAK (Y397) (#8556; Cell Signaling), phospho-FAK (Y925) (#sc-11 766; Santa Cruz), phospho-ERK (p44/42) (#9101; Cell Signaling), Pxn (ab32084; Abcam), FAK (#1688; Santa Cruz), ERK (#9102; Cell Signaling), Tbx1 (ab18530; Abcam), PIP5K1c (ab109192; Abcam), talin (SAB4200694; SigmaAldrich), GAPDH (ab125247; Abcam), lamin B1 (ab133741; Abcam).

Techniques: Control, Microscopy, Transfection, Construct, Activity Assay, Pull Down Assay, Western Blot

Journal: The FASEB Journal

Article Title: Focal Adhesion Kinase Orchestrates GLUT4 Translocation and Glucose Uptake via Cytoskeletal Turnover in Primary Adipocytes

doi: 10.1096/fj.202402764RR

Figure Lengend Snippet: (A) Quantification of FAK Y397 phosphorylation (western blotting) in primary, inguinal mouse adipocytes after 1 h pre‐incubation with respective doses of FAKi (or DMSO, control) and subsequent incubation with (30 min, 10 nM) or without insulin; data shown as mean ± SEM and n = 3–4. (B) Representative western blots of FAK Y397 and total FAK. Heat shock protein 90 (HSP90) was used as the loading control. (C) Glucose uptake in primary, inguinal mouse or (D) primary human, subcutaneous adipocytes after 1 h pre‐incubation with respective doses of FAKi and subsequent incubation with (30 min, 10 nM) or without insulin; n = 3–5; data shown as mean ± SEM. Data in (A) and (C) were analyzed using mixed effects analysis, including a random subject effect (mouse) and fixed effects of stimulation (insulin) and condition (FAKi). Interaction effects of insulin and FAK inhibitor (FAKi × Insulin) were evaluated. (D) was analyzed using a repeated‐measures two‐way ANOVA. The effects of insulin, FAKi, and FAKi × Insulin were investigated. Bonferroni multiple comparison adjustment ( α = 0.05) was used in the post hoc analysis of (A, C, and D). Insulin‐stimulated conditions are displayed in blue. The glucose uptake outcome in (C) was log‐transformed for statistical analysis.

Article Snippet: FAK Y397 , Cell Signaling , 8556 , RRID:AB_10891442 , 1:500 , WB.

Techniques: Phospho-proteomics, Western Blot, Incubation, Control, Comparison, Transformation Assay

Journal: The FASEB Journal

Article Title: Focal Adhesion Kinase Orchestrates GLUT4 Translocation and Glucose Uptake via Cytoskeletal Turnover in Primary Adipocytes

doi: 10.1096/fj.202402764RR

Figure Lengend Snippet: (A) Representative western blots of insulin signaling targets in primary, inguinal mouse adipocytes after 1 h pre‐treatment with 10 μM FAKi and subsequent incubation with (5 min, 10 nM) or without insulin. (B) Corresponding quantifications of FAK Y397 ( n = 3), IRS‐1 Y612 ( n = 3), PKB S473/T308 ( n = 3), AS160 T642 ( n = 3) and ERK1/2 T202/Y204 ( n = 6) phosphorylation; data shown as mean ± SEM. Statistical analysis was performed using two‐way repeated measures ANOVA with Bonferroni multiple comparison adjustment ( α = 0.05). (C) Representative western blots of FAK Y397, PKB S473, AS160 T642, and ERK1/2 T202/Y204 in primary human subcutaneous adipocytes after 1 h pre‐treatment with 10 μM FAKi and subsequent incubation with (5 min, 10 nM) or without insulin; n = 1.

Article Snippet: FAK Y397 , Cell Signaling , 8556 , RRID:AB_10891442 , 1:500 , WB.

Techniques: Western Blot, Incubation, Phospho-proteomics, Comparison

Journal: The FASEB Journal

Article Title: Focal Adhesion Kinase Orchestrates GLUT4 Translocation and Glucose Uptake via Cytoskeletal Turnover in Primary Adipocytes

doi: 10.1096/fj.202402764rr

Figure Lengend Snippet: FIGURE 1 | (A) Quantification of FAK Y397 phosphorylation (western blotting) in primary, inguinal mouse adipocytes after 1 h pre-incubation with respective doses of FAKi (or DMSO, control) and subsequent incubation with (30 min, 10 nM) or without insulin; data shown as mean ± SEM and n = 3–4. (B) Representative western blots of FAK Y397 and total FAK. Heat shock protein 90 (HSP90) was used as the loading control. (C) Glucose uptake in primary, inguinal mouse or (D) primary human, subcutaneous adipocytes after 1 h pre-incubation with respective doses of FAKi and sub- sequent incubation with (30 min, 10 nM) or without insulin; n = 3–5; data shown as mean ± SEM. Data in (A) and (C) were analyzed using mixed ef- fects analysis, including a random subject effect (mouse) and fixed effects of stimulation (insulin) and condition (FAKi). Interaction effects of insulin and FAK inhibitor (FAKi × Insulin) were evaluated. (D) was analyzed using a repeated-measures two-way ANOVA. The effects of insulin, FAKi, and FAKi × Insulin were investigated. Bonferroni multiple comparison adjustment (α = 0.05) was used in the post hoc analysis of (A, C, and D). Insulin- stimulated conditions are displayed in blue. The glucose uptake outcome in (C) was log-transformed for statistical analysis.

Article Snippet: Target Supplier Article No. RRID Dilution Notes AS160 Cell Signaling 2670 RRID:AB_2199375 1:1000 WB AS160 T642 Cell Signaling 4288 RRID:AB_10545274 1:500 WB FAK Cell Signaling 13 009 RRID:AB_2798086 1:1000 WB FAK Cell Signaling 3285 RRID:AB_2269034 1:50 IP FAK Y397 Cell Signaling 8556 RRID:AB_10891442 1:500 WB HSP90 BD Biosciences 610 418 RRID:AB_397798 1:2000 WB PKB S473 Cell Signaling 4060 RRID:AB_2315049 1:1000 WB PKB Cell Signaling 4691 RRID:AB_915783 1:1000 WB IRS- 1 Cell Signaling 3407 RRID:AB_2127860 1:250 WB IRS- 1 Y612 Thermo Fisher 44- 816G RRID:AB_1501247 1:500 WB ERK1/2 T202/Y204 Cell Signaling 4370 RRID:AB_2315112 1:1000 WB Phospho- PAK1 (T423)/PAK2 (T402) Cell Signaling 2601 RRID:AB_330220 1:1000 WB Arp2 (phospho T237 + T238) Abcam ab119766 RRID:AB_10900743 1:250 WB Phospho- Cofilin (S3) Cell Signaling 3313 RRID:AB_2080597 1:500 WB LM048 Integral Molecular CSB0148 RRID:AB_3106913 1:300 CM α- Tubulin Sigma- Aldrich CP06 RRID:AB_2617116 1:300 CM 15306860, 2025, 10, D ow nloaded from https://faseb.onlinelibrary.w iley.com /doi/10.1096/fj.202402764R R by N at Prov Indonesia, W iley O nline L ibrary on [22/05/2025].

Techniques: Phospho-proteomics, Western Blot, Incubation, Control, Comparison, Transformation Assay

Journal: The FASEB Journal

Article Title: Focal Adhesion Kinase Orchestrates GLUT4 Translocation and Glucose Uptake via Cytoskeletal Turnover in Primary Adipocytes

doi: 10.1096/fj.202402764rr

Figure Lengend Snippet: FIGURE 2 | (A) Representative western blots of insulin signaling targets in primary, inguinal mouse adipocytes after 1 h pre-treatment with 10 μM FAKi and subsequent incubation with (5 min, 10 nM) or without insulin. (B) Corresponding quantifications of FAK Y397 (n = 3), IRS-1 Y612 (n = 3), PKB S473/T308 (n = 3), AS160 T642 (n = 3) and ERK1/2 T202/Y204 (n = 6) phosphorylation; data shown as mean ± SEM. Statistical analysis was performed using two-way repeated measures ANOVA with Bonferroni multiple comparison adjustment (α = 0.05). (C) Representative western blots of FAK Y397, PKB S473, AS160 T642, and ERK1/2 T202/Y204 in primary human subcutaneous adipocytes after 1 h pre-treatment with 10 μM FAKi and subsequent incubation with (5 min, 10 nM) or without insulin; n = 1.

Article Snippet: Target Supplier Article No. RRID Dilution Notes AS160 Cell Signaling 2670 RRID:AB_2199375 1:1000 WB AS160 T642 Cell Signaling 4288 RRID:AB_10545274 1:500 WB FAK Cell Signaling 13 009 RRID:AB_2798086 1:1000 WB FAK Cell Signaling 3285 RRID:AB_2269034 1:50 IP FAK Y397 Cell Signaling 8556 RRID:AB_10891442 1:500 WB HSP90 BD Biosciences 610 418 RRID:AB_397798 1:2000 WB PKB S473 Cell Signaling 4060 RRID:AB_2315049 1:1000 WB PKB Cell Signaling 4691 RRID:AB_915783 1:1000 WB IRS- 1 Cell Signaling 3407 RRID:AB_2127860 1:250 WB IRS- 1 Y612 Thermo Fisher 44- 816G RRID:AB_1501247 1:500 WB ERK1/2 T202/Y204 Cell Signaling 4370 RRID:AB_2315112 1:1000 WB Phospho- PAK1 (T423)/PAK2 (T402) Cell Signaling 2601 RRID:AB_330220 1:1000 WB Arp2 (phospho T237 + T238) Abcam ab119766 RRID:AB_10900743 1:250 WB Phospho- Cofilin (S3) Cell Signaling 3313 RRID:AB_2080597 1:500 WB LM048 Integral Molecular CSB0148 RRID:AB_3106913 1:300 CM α- Tubulin Sigma- Aldrich CP06 RRID:AB_2617116 1:300 CM 15306860, 2025, 10, D ow nloaded from https://faseb.onlinelibrary.w iley.com /doi/10.1096/fj.202402764R R by N at Prov Indonesia, W iley O nline L ibrary on [22/05/2025].

Techniques: Western Blot, Incubation, Phospho-proteomics, Comparison