Journal: FEBS Open Bio

Article Title: The p75 NTR and its carboxyl‐terminal fragment exert opposing effects on melanoma cell proliferation and apoptosis via modulation of the NF‐κB pathway

doi: 10.1002/2211-5463.13047

Figure Lengend Snippet: Activation of NF‐κB signaling pathway by p75 NTR ‐CTF. (A) Subcellular localization of NF‐κB p65 (red) was detected by immunofluorescence. Nuclei were stained with DAPI (blue). Scale bar: 100 μm. (B) Cell lysates were extracted from cytosolic and nuclear fractions; the expression levels of p65, β‐actin (cytosolic control) and Lamin B1 (nuclear control) were analyzed by immunoblot analysis. (C) Phosphorylation and total levels of IκBα were assessed by immunoblot analysis. (D) mRNA levels of the antiapoptosis ( c‐IAP1/2 , FLIP ) and tumor angiogenesis ( IL8 , VEGF and bFGF ) were detected by qRT‐PCR. (E) Protein levels of c‐IAP2 and bFGF were measured by western blot analysis. (F, G) Protein levels of IL‐8 and VEGF in the culture media of the cells were measured by ELISA. Data represent the mean ± SD of three separate experiments. Differences between two groups were compared using Student's t ‐test. * P < 0.05; ** P < 0.01; *** P < 0.001. Scale bar: 100 μm.

Article Snippet: The primary antibodies used were p75 NTR (#ab52987; Abcam, Cambridge, UK), NF‐κB p65 (#8242; CST, Danvers, MA, USA), IκBα (#9242; CST, Danvers, MA, USA), Phospho‐IκBα (#2859; CST, Danvers, MA, USA), caspase‐3 (ab214430; Abcam, Cambridge, UK), caspase‐9 (ab202068; Abcam, Cambridge, UK), Bax (ab32503; Abcam, Cambridge, UK), Bcl‐2 (ab32124; Abcam, Cambridge, UK), Lamin B1 (Beyotime, Shanghai, China), inhibitor of apoptosis protein 2 (c‐IAP2; #380798; ZEN BIO, Chengdu, China), bFGF (#381676; ZEN BIO, Chengdu, China) and β‐actin (Sino Biological, Beijing, China). β‐Actin or Lamin B1 expression served as an internal control.

Techniques: Activation Assay, Immunofluorescence, Staining, Expressing, Western Blot, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Heliyon

Article Title: Insulin attenuates apoptosis in neuronal cells by an integrin-linked kinase-dependent mechanism

doi: 10.1016/j.heliyon.2019.e02294

Figure Lengend Snippet: Insulin increases IAP expression in an ECM-dependent manner in Cerebellar Granule Neurons: Inhibition by the ILK Inhibitor KP-392. Representative Western blots of lysates from cerebellar cultures grown on poly-D-lysine or laminin and exposed to serum and depolarization or serum and depolarization withdrawal alone or in the presence of insulin (10 μg/ml) with or without KP-392 (100 μM) for 24 h (n = 2–3). Immunoblots were probed with an antibody against c-IAP2. Bar graph represents densitometric analysis of 3 independent trials of cells grown on laminin, averaged and normalized to cultures subject to serum and depolarization withdrawal. Data are mean ± SEM. *P < 0.05, different from insulin treated group as determined by a Student's t-test. Full, non-adjusted images are not available as supplementary material for Fig. 5 .

Article Snippet: Lysates from primary cultures were prepared in Tris-HCl buffer, pH 7.6 containing 1% NP-40, 150 mM NaCl, 1mM EDTA, 3.8 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM PMSF, 2 mM NaF, and 1 mM Na 3 VO 4 as previously described . c-IAP2 was detected using a rabbit anti-c-IAP2 antibody (a kind gift from Dr. Robert Korneluk, Aegera Therapeutics Inc., QC, CA), ILK was detected using a monoclonal anti-ILK antibody (BD Transduction Laboratories, San Jose, CA), caspase was detected using a monoclonal antibody caspase-3 p11 (Santa Cruz, ON, CA) and GAPDH was detected using a rabbit anti-GAPDH (Santa Cruz, ON, CA).

Techniques: Expressing, Inhibition, Western Blot