rabbit polyclonal anti ire1a  (Thermo Fisher)

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: iScience

Article Title: Type 2 diabetes sex-specific effects associated with E167K coding variant in TM6SF2 .

doi: 10.1016/j.isci.2021.103196

Figure Lengend Snippet: Figure 4. Impaired IRE1a-XBP1 signaling in male Tm6sf2 KI mice were fed an HFD (A) TM6SF2 subcellular localization was measured by immunofluorescence of TM6SF2 fused with GFP protein (TM6SF2- GFP) and TM6SF2-GFP containing E167K variant. After transfection of TM6SF2 expression vector, Huh-7 cells were treated with ethanol (control) or oleic acid (OA, 400 mM) for 18 h. Colocalization was quantified by Pearson’s correlation coefficient (PCC). KDEL, an ER C-terminal tetrapeptide retention signal (Lys-Asp-Glu-Leu), was used an ER marker. Scale bar represents 20 mm. (B and C) The male Tm6sf2 KI mice and (D and E) female Tm6sf2 KI mice were fed an HFD. (B) and (D) Total and phosphorylated IRE1a in the liver was determined by western blot (n = 5/group). The band intensity of phosphorylated IRE1a was analyzed quantitatively and normalized to total IRE1a. (C) and (E) Unspliced X-box-binding protein 1 (XBP1u)

Article Snippet: Huh-7 cells were harvested and used for Co-IP with a rabbit polyclonal anti-IRE1a antibody (Cell Signaling, #3294), followed by immunoblotting with the anti-TM6SF2 antibody to detect the association between IRE1a and TM6SF2 in Huh-7 cells under the normal or lipid overload conditions.

Techniques: Variant Assay, Transfection, Expressing, Plasmid Preparation, Control, Marker, Western Blot, Binding Assay