Journal: Oncotarget

Article Title: Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance

doi: 10.18632/oncotarget.23186

Figure Lengend Snippet: (A) SDF-1α stimulated release of calcium, and number of responding cells, was higher in the presence of PRL-3. (B) CSK stimulated calcium release was independent of PRL-3 expression. Gap indicates time used for removal of tube for adding stimulus. Background signal was recorded for 20 seconds, the sample was then removed and stimulated with SDF-1ɑ (100 ng/mL) or CSK (1:500), and returned for continuing recording for total of 120 seconds. (C) SDF-1α increased the phosphorylation of FAK Tyr 576/577 in Reh shCTRL. Phosphorylation of Src was also increased in Reh shCTRL compared to PRL-3 knockdown cells, but was not induced by SDF-1α stimulation. Total Src and FAK was unchanged by PRL-3 knockdown. The membranes were reprobed for total Src or FAK, and GAPDH. (D) Src inhibition removed the PRL-3 effect of SDF-1ɑ (75 ng/mL) induced migration. Bars represent the percentage of migrated cells from one representative of three independent experiments. Error bars represent + 1 SD of three repeated counts in two parallels. *** indicates p < 0,001.

Article Snippet: Antibody against GAPDH was from Abcam (Cambridge, United Kingdom), and antibodies against phospho-focal adhesion kinase (pFAK) (Tyr576/577) (#3281), total FAK (#3285), pSrc (Y416) (#2101) and total Src (#2109) were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Inhibition, Migration

Journal: Pharmaceutics

Article Title: Anti-Survival Effect of SI306 and Its Derivatives on Human Glioblastoma Cells

doi: 10.3390/pharmaceutics14071399

Figure Lengend Snippet: SI306 derivatives inhibit Src activity in U87 cells. Src inactivation was evaluated by immunoblot analyses of p-Src (Tyr530 and Tyr419) and total Src. The expression levels of p-Src and Src were evaluated in CAS-1 (left panels) and U87 (right panels) cells exposed to 4 µM and 10 µM of compound 1 (SI306, C1), compound 2 (C2), and compound 3 (C3) for 24 h ( a ) and 48 h ( b ). Immunoblots shown are representative of three independent experiments. GAPDH is the internal loading control. Histograms summarize quantitative data of means ± S.E.M. of three independent experiments. Statistical significance of differences was determined by ANOVA followed by Tukey’s test. Data are expressed as a ratio of the levels of p-Src to the levels of total Src. ° p < 0.05 vs. untreated cells; °° p < 0.01 vs. untreated cells; and °°° p < 0.001 vs. untreated cells.

Article Snippet: Immunoblots were performed according to standard methods [ ] using rabbit antibody anti-human anti-Src (#2109), anti-Phospho-Src (Tyr527, #2105) corresponding in humans to tyrosine 530 residue, anti-Phospho-Src (Tyr416, #2101) corresponding in humans to tyrosine 419 residue, anti PARP (#9542), anti-EGFR (#4267), and anti-EGFR-vIII (#2232; Cell Signaling Technology Inc., Danvers, MA, USA, Upstate, Lake Placid, NY, USA).

Techniques: Activity Assay, Western Blot, Expressing

Journal: Nature Communications

Article Title: Disruption of FOXO3a-miRNA feedback inhibition of IGF2/IGF-1R/IRS1 signaling confers Herceptin resistance in HER2-positive breast cancer

doi: 10.1038/s41467-021-23052-9

Figure Lengend Snippet: a The expression of PPP3CB, p-STAT6, STAT6, p-Src, Src, and β-actin in the indicated cells was analyzed by western blot assays (left). The expression levels of PPP3CB mRNA were measured by qRT-PCR (right), **** p < 0.0001. b Pool2 or HR20 cells with PPP3CB overexpression were treated with Herceptin for 72 h (top). SKBR3 or BT474 cells with specific knockdown of PPP3CB were treated with Herceptin for 72 h (bottom). Cell viability was evaluated by MTS assays, SKBR3 sh-1#: ** p = 0.008, ** p = 0.0013, *** p = 0.0002, sh-2#: ** p = 0.0019, **** p < 0.0001, BT474 sh-1#: ** p = 0.0038, *** p = 0.0002, *** p = 0.0001, sh-2#: *** p = 0.0003, *** p = 0.0001, **** p < 0.0001. c Pool2 or HR20 cells were transfected with control vector (Control) or PPP3CB-overexpressing vector (PPP3CB). The expression of PPP3CB, p-FOXO3a, FOXO3a, IRS1, and β-actin was examined by western blot assays (left). IGF2 levels in the CM were detected by ELISA (right). d The expression levels of miR-128-3p, miR-30a-5p, and miR-193a-5p in PPP3CB-overexpressing pool2 or HR20 cells were measured by qRT-PCR, **** p < 0.0001. e Pool2 or HR20 cells were treated with vehicle (Mock) or entinostat (1 μM, Ent.) for 48 h. The expression of PPP3CB, p-FOXO3a, FOXO3a, IRS1, and β-actin was examined by western blots. f , g Pool2 or HR20 cells were transfected with control shRNA (Con or sh-Con) or specific STAT6 shRNAs (sh-2# or sh-4#). The expression of STAT6, PPP3CB, p-FOXO3a, FOXO3a, IRS1, and β-actin was examined by western blots ( f ); the enrichment of HDAC1 at PPP3CB promoter was determined by ChIP-qPCR ( g ), **** p < 0.0001. h Total protein extracts of inducated cells were subjected to IP using an anti-HDAC1 antibody or control IgG, followed by western blot analysis of HDAC1 or p-STAT6. i Pool2 or HR20 cells treated with vehicle (Mock) or SU6656 (10 μM) were examined by western blot analysis. n = 3 biological independent samples ( a , b , c , d , g ). Data are presented as mean values ± SEM ( a , b , c , d , g ). Statistical significance was determined by a two-tailed Student’s t -test ( a , b , c , d , g ). All data are provided in the Source Data.

Article Snippet: Primary antibodies used in western blot assays: IRS1 (#2382, 1:1000), IGF-1R (#3018, 1:1000), p-IGF-1R(Tyr1131)/IR(Tyr1146) (#80732, 1:1000), Akt (#4691, 1:1000), p-Akt(Thr308) (#13038, 1:1000), p-Akt(Ser473) (#4060, 1:1000), S6K (#2708, 1:1000), p-S6K(Thr389) (#97596, 1:1000), FOXO3a (#12829, 1:1000), p-FOXO3a(Ser253) (#9466, 1:1000), STAT6 (#5397, 1:1000), p-STAT6(Tyr641) (#56554, 1:1000), Src (#2109, 1:1000), p-Src(Tyr416) (#59548, 1:1000), and HDAC1 (#34589, 1:1000) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Over Expression, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, shRNA, Two Tailed Test

Journal: Nutrients

Article Title: KYMASIN UP Natural Product Inhibits Osteoclastogenesis and Improves Osteoblast Activity by Modulating Src and p38 MAPK

doi: 10.3390/nu14153053

Figure Lengend Snippet: List of primary and secondary antibodies used in WB.

Article Snippet: Rabbit monoclonal anti-Src (36D10) , 60 , 1:5000 , Cell Signaling Technol., Danvers, MA, USA.

Techniques: