Journal: eLife

Article Title: B cell receptor-induced IL-10 production from neonatal mouse CD19 + CD43 - cells depends on STAT5-mediated IL-6 secretion

doi: 10.7554/eLife.83561

Figure Lengend Snippet: ( A ) CD19 + CD43 - cells were stimulated with anti-IgM antibodies for the indicated duration and changes in PKC (Ser 660 ), FAK (Tyr 397 ), and FAK (Tyr 567 ) phosphorylations were detected in western blot analysis (biological triplicate). ( B ) CD19 + CD43 - cells were pre-treated with DMSO, 20 nM PKC inhibitor Staurosporine (Staur), or 10 μM FAK inhibitor 14 (F-14) for 1 hr and then stimulated with anti-IgM antibodies for the indicated duration. Changes in FAK (Tyr 397 ) and STAT5 (Tyr 694/699 ) phosphorylations were detected in Western blot analysis (biological triplicate). ( C ) Isolated CD19 + CD43 - B cells were pre-treated with DMSO, 20 nM Staurosporine, or 10 μM F-14, or 20 μM Pimozide for 1 hr prior to incubation in the absence or presence of anti-IgM antibodies for 4 hr and Il6 mRNA expression was determined by RT-qPCR (biological triplicate). ( D ) CD19 + CD43 - cells were pre-treated with Rac1 inhibitor NSC23766 for 1 hr and then stimulated with anti-IgM antibodies for 15 min. Changes in STAT5 (Tyr 694/699 ) phosphorylation was detected in western blot analysis (biological triplicate). ( E ) Isolated CD19 + CD43 - B cells were pre-treated with NSC23766 for 1 hr prior to incubation in the absence or presence of anti-IgM antibodies for 20 hr and Il6 mRNA expression was determined by RT-qPCR (biological triplicate). In all experiments, data shown as the mean  ± s.d. of three biological replicates. P values were calculated using one-way ANOVA with a Dunnett’s multiple comparisons test (*p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001). Figure 6—source data 1. Raw data of all western blots from . Figure 6—source data 2. Complete and uncropped membrane of all western blots from .

Article Snippet: The following antibodies used in blotting were from Cell Signaling Technology (Danvers, MA): anti-phospho STAT1 Tyr 701 (58D6), anti-STAT1 (42H3), anti-phospho STAT3 Tyr 706 (D3A7), anti-STAT3 (79D7), anti-phospho STAT5 Tyr 694 (D47E7), anti-STAT5 (D2O6Y), anti-beta-actin (D6A8), anti-phospho PKC (pan) betaII Ser 660 , anti-phospho FAK Tyr 397 , anti-FAK, anti-phospho Akt Ser 473 (D9E), anti-Akt (pan) (C67E7), anti-phospho p38 MAPK Thr 180 /Tyr 182 (D3F9), anti-p38 MAPK (D13E1), anti-phospho SAPK/JNK Thr 183 /Tyr 185 (81E11), anti-SAPK/JNK, anti-phospho p44/42 MAPK (Erk1/2) Thr 202 /Tyr 204 (D13.14.4E), anti-p44/42 MAPK (Erk1/2) (137F5), anti-phospho NF-κB p65 Ser 536 (93H1), anti-NF-κB p65 (D14E12), anti-phospho IκBα Ser 32 (14D4), anti-IκBα (44D4), anti-rabbit IgG, HRP-linked.

Techniques: Western Blot, Isolation, Incubation, Expressing, Quantitative RT-PCR

Journal: BMC Research Notes

Article Title: Optimising parameters for the differentiation of SH-SY5Y cells to study cell adhesion and cell migration

doi: 10.1186/1756-0500-6-366

Figure Lengend Snippet: (a) Immunofluorescent staining of activated FAK in SH-SY5Y cells. SH-SY5Y cells were plated on laminin coated coverslips in complete DMEM (undifferentiated) or differentiated in serum free DMEM with IGF-1 for 72 hours. Cells were fixed in 4% paraformaldehyde, permeabilised in PHEM/0.1% Triton X and blocked in PHEM/5% goat serum. Cells were stained with either pFAK Y 397 or pFAK Y 925 (red) and the nuclei were stained with Hoechst 33242 (blue). All images were acquired sequentially at 63× and images merged using ImageJ. Scale bar = 20 μm. (b) FAK was immunoprecipitated from lysates of SH-SY5Y cells, undifferentiated or differentiated with IGF-1 and run on 12% SDS-PAGE gels and probed for phospho-tyrosine and FAK, followed by detection with LI-COR Odyssey™. Densitometry of protein bands was measured using LI-COR Odyssey™ software and normalised to undifferentiated levels. The fold increase in pFAK signal compared to undifferentiated protein level are plotted on a bar chart ± SEM, n = 3.

Article Snippet: Anti-phospho FAK (Tyr-925) polyclonal antibody was from Cell Signalling Technology (Beverly, MA).

Techniques: Staining, Immunoprecipitation, SDS Page, Software

Journal: Journal of Translational Medicine

Article Title: An aberrant spliced transcript of focal adhesion kinase is exclusively expressed in human breast cancer

doi: 10.1186/1479-5876-12-136

Figure Lengend Snippet: Detection of FAK transcripts in human breast tumor tissues. A . Schematic of the human FAK molecular domains. The wild-type FAK contains a FERM domain (1–415 amino acid), a kinase domain (416–676 amino acid), and a C-terminus segment (677–1052 amino acid) that includes two proline-rich regions and a FAT domain ( a ). Three Tyr phosphorylation sites, i.e., Tyr 397, 576, and 577, were found in both wild-type FAK ( a ) and the protein encoded by -26-exon FAK ( b ), but the latter lacks the segment composed of amino acids 744 to 789, which contains the caspase-3/-7-like cleavage motif (DQTD) and has a mutation (Leucine mutated to Proline) at the C-terminus ( b ). B . Occurrence frequency of -26-exon FAK in normal and breast tumor tissues. The -26-exon FAK is exclusively expressed in tumor tissues. C . Agarose gel electrophoresis was used to analyze the pattern of FAK gene transcripts in normal and breast tumor tissues. The short splicing product of FAK (-26-exon FAK) was only observed in tumor tissues. M: marker; N: normal tissue; T: tumor tissue. D . Schematic of the strategy to determine the percentages of -26-exon FAK expression in tumor samples. E . The percentage of -26-exon FAK expression in tumor samples. The tumor sample 7 and sample 8 did not express the -26-exon FAK. The percentage of -26-exon FAK expression in tumor samples 1 ~ 6 was 30% ~ 90%. F . Examination of the -26-exon FAK expression in tumor samples by western blot. The tumor samples 1 ~ 5 expressing the -26-exon FAK and the corresponding normal samples were analyzed by western blot using the anti-FAK antibody. N: normal tissue; T: tumor tissue.

Article Snippet: The following antibodies were used in this study: anti-FAK (BD Biosciences, 1:2000 dilution), anti-Tyr-397 (Cell Signaling, 1:1000 dilution), anti-Tyr-576/577 (Cell Signaling, 1:1000 dilution), anti-caspase-3 (Cell Signaling, 1:1000 dilution), and anti-caspase-7 (Cell Signaling, 1:1000 dilution).

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Marker, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Pharmacological Inhibition of FAK-Pyk2 Pathway Protects Against Organ Damage and Prolongs the Survival of Septic Mice

doi: 10.3389/fimmu.2022.837180

Figure Lengend Snippet: Effect of PF271 on tissue activation of FAK-PyK2 pathway during experimental sepsis. Mice were randomly selected to undergo Sham or CLP surgery. One hour later, CLP mice were treated once with either Vehicle or PF271 (25 mg/kg s.c.). Twenty-four hours after Sham or CLP procedure, liver and kidney were harvested, and the total protein was extracted from them. Western blotting analysis for phosphorylation of Tyr 397 on FAK in the liver (A) and kidney (B) were normalized to total FAK; Phosphorylation of Tyr 402 on PyK2 in the liver (C) and kidney (D) were normalized to total PyK2; Phosphorylation of Thr 180 /Tyr 182 on p38 in the liver (E) and kidney (F) were normalized to total p38. Densitometric analysis of the bands is expressed as relative optical density (O.D.). Data are expressed as dot plots for each animal and as mean ± SD of 4-5 mice per group. Statistical analysis was performed by one-way ANOVA followed by Bonferroni’s post hoc test. *p < 0.05 CLP vs Sham/CLP+PF271.

Article Snippet: The antibodies used were: rabbit anti-Thr 180 /anti-Tyr 182 p38 (Cell Signaling #9211); rabbit anti-total p38 (Cell Signaling #9212); mouse anti-NRLP3 (Adipogen- AG-20B-0014-C100); rabbit anti-Caspase-1 (Cell Signaling #24232); rabbit anti-Tyr 397 FAK (Cell Signaling #3283); rabbit anti-total FAK (Cell Signaling #3285); mouse anti-Tyr 402 PyK2 (Cell Signaling #3291); mouse anti-total PyK2 (Santa Cruz #sc-393181); rabbit anti-β-actin (Cell Signaling #4970).

Techniques: Activation Assay, Western Blot