rabbit anti phospho ire1α novus nb100 2323 rabbit polyclonal antisera ribophorin  (Novus Biologicals)

Journal: Lipids in health and disease

Article Title: GRP78/BiP alleviates oxLDL-induced hepatotoxicity in familial hypercholesterolemia caused by missense variants of LDLR in a HepG2 cellular model.

doi: 10.1186/s12944-023-01835-x

Figure Lengend Snippet: Fig. 6 A graphical abstract summarizing the study model and major findings. The thin black arrows and red lightning symbols represent ER stress-caused by ER retention of LDLR, and oxLDL induced stress- caused by oxidation of excess levels of LDL. The brown, maroon and blue colored arrows represent ER stress dissipated along the ATF6, PERK and IRE1A branches, respectively. The pacman symbol denotes S1P/S2P protease in the Golgi Complex. The red inhibitory arrows denote the proteins that inhibit/are inhibited during the process. The scissors symbolize splicing of XBP-1 transcripts. The blue dots and red arrows in the mitochondria denote release of cytochrome C caused by disrupted mitochondrial membrane potential. Purple arrows-direction of LDH release from cells due to cytotoxicity. The continuous low-medium dial denotes restored cellular responses brought about by neon transfection of GRP78/BiP plasmid along (A) ER, (B) mitochondria and (C) plasma membrane. The downward arrows denote lowering of target proteins evaluated

Article Snippet: The antibodies used and their dilutions are as follows: Rabbit polyclonal anti-Alpha Tubulin (1 in 1000, Cell Signaling Technology, Cat. No. 2144S), Rabbit polyclonal anti-GAPDH (1 in 1000, Cell Signaling Technology, Cat. No. 2118S), Rabbit polyclonal anti-FLAG (1 in 1000, Cell Signaling Technology, Cat. No. 2368S), Rabbit polyclonal anti-IRE1A (1 in 500, Cell Signaling Technology, Cat. No. 3214S), Rabbit polyclonal phospho-IRE1A (Ser724) (1 in 500, Novus Biologicals, NB100-2323), Rabbit polyclonal anti-eIF2A (1 in 500, Cell Signaling Technology, Cat. No. 9722S), Rabbit polyclonal anti phospho-eIF2A (Ser51) (1 in 500, Cell Signaling Technology, Cat. No. 9721S), Rabbit polyclonal anti-XBP1 Antibody (1 in 1000, Novus Biologicals, NB2-20,917), Rabbit polyclonal anti-GRP78/BiP (1 in 1000, Cell Signaling Technology, Cat. No. 3177S), Rabbit polyclonal antiBIM (1 in 300, Novus Biologicals, NBP2-67,456), Rabbit polyclonal anti-phospho BIM (1 in 300, Cell Signaling Technology, Cat. No. 12433S), Mouse polyclonal antiCHOP (1 in 250, Cell Signaling Technology, Cat. No. 2895S), Rabbit polyclonal total Caspase 3 antibody (1 in 1000, Cell Signaling Technology, Cat. No. 9662S), Rabbit polyclonal anti-cleaved caspase 3 (1 in 250, Cell Signaling Technology, Cat. No. 9664S), Mouse monoclonal antiJNK (1 in 500, Cell Signaling Technology, Cat. No. 3708), Rabbit polyclonal anti-phospho JNK (Thr183/Tyr 185) (1 in 250, Cell Signaling Technology, Cat. No.9251), Mouse monoclonal anti-Cytochrome c antibody (1 in 300, Thermo Fisher Scientific, Cat. No. MA5-11,674), Goatraised, HRP-conjugated secondary antibodies against Mouse (1 in 30,000, Cat. No. 115–035-1661) and Rabbit (1 in 30,000, Cat. No. 111–035-1441) were purchased from Jacksons ImmunoResearch Europe LTD. Blots were quantitatively analysed by densitometric analysis of respective signal intensities normalized to Alpha tubulin or GAPDH as loading controls.

Techniques: Membrane, Transfection, Plasmid Preparation, Clinical Proteomics

Journal: The Journal of investigative dermatology

Article Title: Vitiligo inducing phenols activate the unfolded protein response in melanocytes resulting in upregulation of IL6 and IL8

doi: 10.1038/jid.2012.181

Figure Lengend Snippet: (a) Quantitative real-time PCR demonstrates that mRNA levels of IL6 and IL8 are increased in human melanocytes treated with 4-TBP versus controls (average of 4 experiments, two melanocyte lines, * P < 0.001 vs. 0 h). (b) Western blot analysis of cell lysates collected from human melanocytes treated with 4-TBP, MBEH, and thapsigargin (TG) showed increased expression of phosphorylated IRE1. (c) Semi-quantitative RT-PCR of RNA from human melanocytes treated with 4-TBP, MBEH, and TG showed increased splicing of XBP1 compared with control cells. (d) Western blotting of concentrated media collected from human melanocytes treated with 4-TBP or MBEH showed increased secretion of IL6 and IL8. Purified IL6 (Santa Cruz Biotechnology, Inc, CA) Cell Signaling, Danvers, MA), was used as a positive control. Three experiments with two different melanocyte lines yielded similar result. Three experiments performed with two different melanocyte lines, from unrelated donors, yielded similar results.

Article Snippet: After blocking with 5% nonfat milk in TBS and 0.1% Tween 20, membranes were incubated overnight at 4° C with the following primary antibodies: mouse monoclonal antibodies for IL6 (1:500) and IL8 (1:500) (Santa Cruz Biotechnology, CA), IRE1 (1:1000) and EIF2α (1:5000) and rabbit monoclonal phospho-EIF2α (1:1000) (Cell Signaling, Danvers, MA), NRF2 (1:2000) (R&D Systems, Inc. Minneapolis, MN), and Rabbit polyclonal phospho-IRE1 (1:1000) (Novus Biologicals, Littleton, CO) and phospho-EIF2α (Cell Signaling, Danvers, MA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Quantitative RT-PCR, Control, Purification, Positive Control