anti-pfak  (Cell Signaling Technology Inc)

Journal: The FEBS journal

Article Title: Cadherin-6 controls neuronal migration during mouse neocortical development via an integrin-mediated pathway.

doi: 10.1111/febs.70150

Figure Lengend Snippet: Fig. 7. KD and overexpression of Cdh6 did not alter the levels of Paxillin protein and phosphorylated AKT. (A) Immunostaining for Paxillin (top, red) or pFAK (red, bottom) and EGFP (green) using sections from the E18.0 cerebral cortex, which were electroporated with control (n = 3) or Cdh6 KD vector (n = 3) (left) or control (CAG empty) (n = 3) or CAG-Cdh6-HA vectors (n = 3) (right) with CAG-EGFP vectors at E14.0. (B) Western blot analysis of neocortical cells electroporated with CAG empty or CAG-Cdh6- HA vectors using antibodies against HA, Paxillin, pFAK, FAK, and GAPDH (a loading control) (n = 3). Sections in (A) were stained with DAPI (magenta). Scale bar: 50 lm in (A). KD, knockdown.

Article Snippet: The primary antibodies used in this study were as follows: CDH6 (sheep; R&D Systems, Minneapolis, MN, USA, AF2715, 1 : 200), HA (rat; Roche, Basel, Switzerland, 3F10, 1 : 500), Nestin (chick; Aves Labs, Davis, CA, USA, #NES, 1 : 400), DCX (rabbit; CST, Danvers, MA, USA, #4604, 1 : 500), RORB (mouse; Perseus Proteomics, Tokyo, Japan, PPN7927-00, 1 : 400), CUX1 (rabbit; Proteintech, Rosemont, IL, USA, 11733-1AP, 1 : 600), CD29 (clone 9EG7) (rat; BD, San Jose, CA, USA, 550531), Paxillin (rabbit; Abcam, Cambridge, MA, USA, ab32084, 1 : 200), pFAK (Y397) (rabbit, CST, 3283S, 1 : 1000).

Techniques: Over Expression, Immunostaining, Control, Plasmid Preparation, Western Blot, Staining, Knockdown

Journal: Cell Death & Disease

Article Title: Role of the PI3K/AKT signaling pathway in the cellular response to Tumor Treating Fields (TTFields)

doi: 10.1038/s41419-025-07546-8

Figure Lengend Snippet: A – C Western blot analysis of AKT, pAKT (Ser473), FAK, pFAK (Tyr397), and GAPDH in lysates from control and TTFields-treated (2 h) A2780 and H1299 cells ( A ). Densitometric analysis for phosphorylation fold change is shown as mean ± SEM; N ≥ 3. * p < 0.05, ** p < 0.01, and *** p < 0.001; multiple unpaired t-tests. Western blot analysis of FAK, pFAK (Tyr397), AKT, pAKT (Ser473), and GAPDH in lysates from control and TTFields-treated (2 h) H1299 cells in the presence of ROCK inhibitor (ROCK i ) ( D ) or FAK inhibitor (FAK i ) ( E ). Densitometric analysis for phosphorylation fold change is shown as the mean ± SEM; N ≥ 3 . * p < 0.05, ** p < 0.01, and *** p < 0.001; one-way ANOVA followed by Tukey’s post hoc test. F - I Western blot analysis of AKT, pAKT (Ser473), BAD, pBAD (Ser136), FAK, pFAK (Tyr397), and GAPDH in lysates from control and TTFields-treated (24, 48, and 72 h) A2780 and H1299 cells. Densitometric analysis for phosphorylation fold change is shown as mean ± SEM; N ≥ 3. * p < 0.05, ** p < 0.01, and *** p < 0.001; one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Electrophoresis was performed on two different gels for each sample, one immunoblotted for total protein levels, and the other for the levels of the phosphorylated protein, using the following primary antibodies: 4E-BP1 (Cell Signaling, 9452), p4E-BP1 (Thr37/46) (Cell Signaling, 9459), AKT (Cell Signaling, 2920), pAKT (Ser473) (Cell Signaling, 4060), pAKT (Thr308) (Cell Signaling, 4056), AMPK (Cell Signaling, 2793), pAMPK (Thr172) (Cell Signaling, 2535), BAD (Abcam, ab32445), pBAD (Ser136) (Cell Signaling, 43665), FAK (Cell Signaling, 32855), pFAK (Tyr397) (Cell Signaling, 32835), GAPDH (Santa Cruz, sc-32233), GEF-H1 (Cell Signaling, 4145), pGEF-H1 (Ser886) (Cell Signaling, 14143), PI3K p85a (Cell Signaling, 13666), pPI3K p85 (Tyr458)/p55 (Tyr199) (Cell Signaling, 4228), N-cadherin (Abcam, ab18203), S6 (Cell Signaling, 4859), pS6 (S235/236) (Cell Signaling, 4858), ULK1 (Cell Signaling, 8054), pULK1 (Ser757) (Cell Signaling, 6888).

Techniques: Western Blot, Control, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Role of the PI3K/AKT signaling pathway in the cellular response to Tumor Treating Fields (TTFields)

doi: 10.1038/s41419-025-07546-8

Figure Lengend Snippet: A Activation through cell-surface interactions. Without treatment, the catalytic activity of GEF-H1 is attenuated by microtubule binding. TTFields exert directional forces on microtubules, leading to their disassembly and hence to the release of GEF-H1, promoting activation of the GEF-H1/RhoA/ROCK signaling pathway, leading to the formation of focal adhesions and activation of the downstream target FAK (1). This, in turn, leads to activation of the PI3K/AKT axis, with downstream inactivation of the pro-apoptotic protein BAD and subsequent cell survival (2). Co-application of TTFields with an inhibitor of the PI3K/AKT pathway blocks cell survival signals, sensitizing tumor cells to TTFields-induced cell death (3). B Activation through cell-cell interactions. Without treatment, N-cadherin forms adherens junctions between adjacent cells and triggers PI3K/AKT signaling. Exposure to TTFields potentiates multiple homophilic ligations of N-cadherin, thus elevating intercellular N-cadherin interactions (1), increasing PI3K/AKT signal amplitude, and augmenting BAD inactivation, consequently leading to cell survival (2). Co-application of TTFields with an inhibitor of the PI3K/AKT pathway blocks the cell survival signals, sensitizing the tumor cells to TTFields-induced cell death (3). Protein phosphorylation sites: GEF-H1, Ser886; FAK, Tyr397; PI3K p85, Tyr458; AKT, Ser473; BAD, Ser136.

Article Snippet: Electrophoresis was performed on two different gels for each sample, one immunoblotted for total protein levels, and the other for the levels of the phosphorylated protein, using the following primary antibodies: 4E-BP1 (Cell Signaling, 9452), p4E-BP1 (Thr37/46) (Cell Signaling, 9459), AKT (Cell Signaling, 2920), pAKT (Ser473) (Cell Signaling, 4060), pAKT (Thr308) (Cell Signaling, 4056), AMPK (Cell Signaling, 2793), pAMPK (Thr172) (Cell Signaling, 2535), BAD (Abcam, ab32445), pBAD (Ser136) (Cell Signaling, 43665), FAK (Cell Signaling, 32855), pFAK (Tyr397) (Cell Signaling, 32835), GAPDH (Santa Cruz, sc-32233), GEF-H1 (Cell Signaling, 4145), pGEF-H1 (Ser886) (Cell Signaling, 14143), PI3K p85a (Cell Signaling, 13666), pPI3K p85 (Tyr458)/p55 (Tyr199) (Cell Signaling, 4228), N-cadherin (Abcam, ab18203), S6 (Cell Signaling, 4859), pS6 (S235/236) (Cell Signaling, 4858), ULK1 (Cell Signaling, 8054), pULK1 (Ser757) (Cell Signaling, 6888).

Techniques: Activation Assay, Activity Assay, Binding Assay, Phospho-proteomics