Journal: PLoS ONE

Article Title: Insulin Resistance Induced by Hyperinsulinemia Coincides with a Persistent Alteration at the Insulin Receptor Tyrosine Kinase Domain

doi: 10.1371/journal.pone.0108693

Figure Lengend Snippet: Timecourse and insulin dose response of IR phosphorylation at ( A ) pY IR 972 and ( B ) pY IR 1162,1163 detected by western blot with phospho-specific rabbit polyclonal antibodies. Detection and quantification was by infrared immunofluorescence linked to the anti-rabbit secondary antibody. Representative westerns shown for a dose response (right panels; only shown for the shortest insulin incubation period). ( C ) Total IR protein quantified on the same western blots with an anti-IR mouse monoclonal antibody conjugated with an anti-mouse secondary antibody linked to a distinct infrared fluorophore. Total ( D ) tyrosine and ( E ) serine phosphorylation, over all sites on the IR determined by ELISA, in response to insulin stimulation. Data are presented as mean ± sd from 3, 10 and 9 independent studies for the 5 nm, 17 nM and 170 nM doses. The intermediate 45 mins time point was included in a subset of those studies (3, 4 and 4 studies for the 5 nm, 17 nM and 170 nM doses). Since the absolute ‘light unit’ values quantified differ from experiment to experiment, the data were normalized to a treatment condition common amongst all experimental conditions. The earliest time point of induction (2–10′) with 17 nM insulin was set as 1.0 and all other data points within each study were normalized to that. * , increased IR phosphorylation (p<0.05) at the 2–10′ time point relative to the no insulin control. #, diminished IR phosphorylation at longer insulin incubation periods (p<0.05) relative to the 2–10′ time point. The data normalization and statistical symbols are applied similarly to the data presented in the remaining figures.

Article Snippet: Membranes were blocked for 1–2 hr at room temperature in Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) and incubated overnight at 4°C with the following primary antibodies in blocking buffer containing 0.1% Tween-20: mouse monoclonal anti-IR (MA-20, 0.17 μg/ml final concentration), rabbit polyclonal anti-pY972 (Abcam, ab-5678, 1∶4000), rabbit polyclonal anti-pY1162,1163 (Santa Cruz, sc-25103, 1∶500), mouse monoclonal anti-ERK (Cell Signaling Technology, 1∶4000, Beverly, MA, USA), rabbit polyclonal anti-pERK (Cell Signaling Technology, 1∶2000), rabbit polyclonal anti-AKT (Cell Signaling Technology, 1∶2000), rabbit polyclonal anti-pAKT (Cell Signaling Technology, 1∶1000), rabbit polyclonal anti-GSK (Cell Signaling Technology, 1∶1000), and mouse monoclonal anti-pGSK (Cell Signaling Technology, 1∶1000).

Techniques: Phospho-proteomics, Western Blot, Immunofluorescence, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Oncology

Article Title: TIPE2 Suppresses Malignancy of Pancreatic Cancer Through Inhibiting TGFβ1 Mediated Signaling Pathway

doi: 10.3389/fonc.2021.680985

Figure Lengend Snippet: TIPE2 inhibited PI3K/AKT and Raf/MEK/ERK signaling pathways triggered by TGFβ1. (A) The total proteins extracted from cells were analyzed for the expression of AKT, ERK and Bax using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK, anti-Bax and anti-GAPDH. (B) ELISA analysis of TGFβ1 secretion from AsPC-1/vector and AsPC-1/TIPE2 cells. (C) Immunohistochemistry analysis of the TGFβ1 expression in AsPC-1/vector and AsPC-1/TIPE2 tumor tissues. (D) Western blot analysis of the expression of p-TGFBR1 and total TGFBR1 in AsPC-1/vector and AsPC-1/TIPE2 cells. (E) AsPC-1 cells were seeded in 6-well plate and added with or without anti-TGFβ1 antibody or rhTGFβ1 protein. After 48 h incubation, the total proteins extracted from the cultured AsPC-1 cells were analyzed for the expression of AKT and ERK using western blot. The antibodies used were anti-pAKT, anti-AKT, anti-pERK, anti-ERK and anti-GAPDH. Data shown were representative of three independent experiments. Values are presented as means ± SD. ***p < 0.001.

Article Snippet: The primary antibodies include rabbit anti-TIPE2 (ProteinTech), anti-pERK, anti-ERK, anti-pAKT, anti-AKT, anti-Bax (Cell Signaling Technology), anti-GAPDH (ProteinTech), anti-pTGFBR1 and anti-TGFBR1 (Affinity, Cincinnati, OH).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Immunohistochemistry, Incubation, Cell Culture

Journal: Cell metabolism

Article Title: Abrogating mitochondrial dynamics in mouse hearts accelerates mitochondrial senescence

doi: 10.1016/j.cmet.2017.09.023

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyconal anti-PERK , Cell Signaling Technology , Cat# 3192.

Techniques: Virus, Plasmid Preparation, Recombinant, TUNEL Assay, Software