Journal: bioRxiv

Article Title: Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

doi: 10.1101/2024.02.20.581175

Figure Lengend Snippet: A. Correlation of the top 100 upregulated platelet lysate proteins, in response to thrombin, with serum fructosamine. SEC61B was the only platelet protein significantly correlated with serum fructosamine (red), Spearman’s rho = 0.33, p = 0.029. B. Log 2 LFQ of SEC61B in platelets grouped in normal fructosamine (200-290 µmol/L) (blue) and high fructosamine (>290 µmol/L) (red), C. Log 2 LFQ of GRP78 (also known as HSPA5 or BIP) in platelet lysates from patients without (non-DM, blue) or with diabetes (DM, red), t-test with Welch’s correction D. Ratio of phosphorylated IRE-1 (p-IRE1) to beta actin of platelet lysates from patients without (non-DM, blue) or with diabetes (DM, red) detected by Western blot. Mann-Whitney test. E. Platelets stained with antibody against SEC61B (red) and GP1Bb (green) isolated from normoglycemic (Veh) or hyperglycemic (STZ) C57BL/6 mice. Representative images. Scale bar 5 µm. F. Average SEC61B, G. GRP78 (HSPA5, BIP), and H. p-IRE1 fluorescence intensity of immunostained platelets from normoglycemic (Veh, n=3, blue) and hyperglycemic (STZ, n=4-5, red) mice. Average from n=15-20 platelets per mouse. Unpaired t-test with Welch’s correction. GP1Bb: glycoprotein 1B beta; GRP78: glucose regulated protein 78; HSPA5: heat shock protein A5, IRE1: inositol-requiring enzyme 1; Veh = vehicle, STZ = streptozotocin.

Article Snippet: Sections were stained with rabbit anti-phospho-IRE1 (1:100, Invitrogen, Cat#PA5-105424), rabbit anti-SEC61B (1:200, Cell Signaling Technology Cat #14648S), and rabbit anti-GRP78 (1:200 dilution, Abcam, cat# ab21685).

Techniques: Western Blot, MANN-WHITNEY, Staining, Isolation, Fluorescence

Journal: bioRxiv

Article Title: Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

doi: 10.1101/2024.02.20.581175

Figure Lengend Snippet: A. Spearman’s rho correlation of the iBAQ abundance of the B subunit with the A1 and G subunits of the SEC translocon. Log2 LFQ of SEC61A1 and SEC61G in platelet lysate from patients with normal fructosamine (200-290 uM) versus high fructosamine (>290 uM); unpaired t-test with Welch’s correction B. Spearman’s rho correlation of SEC61B abundance with ER proteins HSPA5 (GRP78), CALR, CANX, UGGT1; Platelet lysate Log2 GRP78 LFQ by DM status C. Detection of p-IRE1 in resting lysates from non-DM and DM patients by western blot. Representative images. Detection of p-IRE1 in resting platelets isolated from non-hyperglycemic (Veh) and hyperglycemic (STZ) mice by immunofluorescent staining. Platelet receptor GPIbβ is shown in green and p-IRE1 in red. Representative images. Scale bar 20µm.

Article Snippet: Sections were stained with rabbit anti-phospho-IRE1 (1:100, Invitrogen, Cat#PA5-105424), rabbit anti-SEC61B (1:200, Cell Signaling Technology Cat #14648S), and rabbit anti-GRP78 (1:200 dilution, Abcam, cat# ab21685).

Techniques: Western Blot, Isolation, Staining

Journal: bioRxiv

Article Title: Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

doi: 10.1101/2024.02.20.581175

Figure Lengend Snippet: A. SEC61B fluorescence intensity in the bone marrow of Apoe-/- mice injected with Veh (left panel) or STZ (right panel), stained for SEC61B (red) and GP1bβ (green). Nuclei are stained with Hoescht 33258 (blue). Representative images. Scale bar 20µm. B. Mean megakaryocyte SEC61B fluorescence intensity per area and C. Mean megakaryocyte p-IRE1 fluorescence intensity per area of Veh or STZ treated Apoe-/- mice. D. SEC61B fluorescence intensity in the bone marrow of outbred mice with normoglycemia (left panel) or hyperglycemia (right panel) stained for SEC61B (red) and GP1bβ (green). Nuclei are stained with Hoescht 33258 (blue). Representative images. Scale bar 20µm. E. Mean megakaryocyte SEC61B fluorescence intensity per area and F. Mean megakaryocyte p-IRE1 fluorescence intensity per area of normoglycemic and hyperglycemic mice. n=5 mice for all groups. Mann-Whitney test.

Article Snippet: Sections were stained with rabbit anti-phospho-IRE1 (1:100, Invitrogen, Cat#PA5-105424), rabbit anti-SEC61B (1:200, Cell Signaling Technology Cat #14648S), and rabbit anti-GRP78 (1:200 dilution, Abcam, cat# ab21685).

Techniques: Fluorescence, Injection, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

doi: 10.1101/2024.02.20.581175

Figure Lengend Snippet: A. Detection of p-IRE1 in megakaryocytes (outlined) of Apoe-/- with (STZ) or without (Veh) hyperglycemia and B. outbred mice with or without hyperglycemia by immunofluorescent staining. p-IRE1 is shown in red and nuclei in blue. Representative images. scale bar 20μm C. Immunofluorescence intensity of GRP78 in Apoe-/- mice without hyperglycemia (Veh, blue) versus with hyperglycemia (STZ, red) and D. outbred mice without hyperglycemia (blue) versus with hyperglycemia (red); Mann-Whitney test.

Article Snippet: Sections were stained with rabbit anti-phospho-IRE1 (1:100, Invitrogen, Cat#PA5-105424), rabbit anti-SEC61B (1:200, Cell Signaling Technology Cat #14648S), and rabbit anti-GRP78 (1:200 dilution, Abcam, cat# ab21685).

Techniques: Staining, Immunofluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Identification of SEC61B as a novel regulator of calcium flux and platelet hyperreactivity in diabetes mellitus

doi: 10.1101/2024.02.20.581175

Figure Lengend Snippet: A. ER Calcium efflux is potentiated by puromycin in healthy human platelets. B. Time course of cytosolic calcium changes after TG (2μM, red) and BHQ (10µM, purple) in human platelets (n=3 healthy donors). C. Ratio of phosphorylated IRE-1 (p-IRE1) to GAPDH, D. CD62P and PAC-1, E. Surface PDI and ERp5, F. SEC61B to GAPDH band intensity of platelets treated with Veh (grey), TG (red), or BHQ (purple), Wilcoxon test. BHQ: 2,5,-di-t-butyl-1,4-benzohydroquinone; ERp5: endoplasmic reticulum protein 5; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; IRE1: inositol-requiring enzyme 1; PDI: protein disulfide isomerase; TG: thapsigargin.

Article Snippet: Sections were stained with rabbit anti-phospho-IRE1 (1:100, Invitrogen, Cat#PA5-105424), rabbit anti-SEC61B (1:200, Cell Signaling Technology Cat #14648S), and rabbit anti-GRP78 (1:200 dilution, Abcam, cat# ab21685).

Techniques:

Journal: Cell reports

Article Title: Vitamin K-dependent carboxylation regulates Ca 2+ flux and adaptation to metabolic stress in β cells

doi: 10.1016/j.celrep.2023.112500

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-phospho(S724)-IRE1 , Abcam , Cat# ab124945; RRID:AB_11001365.

Techniques: Recombinant, SYBR Green Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, RNA Sequencing Assay, Expressing, Clone Assay, Sequencing, Knock-Out, Software, CRISPR, Transfection, Purification

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