mouse anti grp75  (Santa Cruz Biotechnology)

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: GRP75 is essential for adipocyte browning. a Five most representative subcellular localization analyses of 63 overlapping proteins. b Representative IHC images and the staining scores of GRP75 (top) in 63 pairs of patient tumours (T) and matched adjacent normal tissues (N). Scale bar, 200 μm. c Kaplan‒Meier survival analysis of patients with ESCC stratified by GRP75 expression ( n = 107; P < 0.001, log-rank test). d Immunoblots of GRP75 in EVs derived from mouse colon cancer cells (MC38 and C26), ESCC cells (YES2 and KYSE150) and other cachexia-inducing tumour cells (HepG2, LLC, AsPC-1, and BxPC3). e MtDNA copy number was detected in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-EVs. f Oxygen consumption rate (OCR) in primary adipocytes treated by GRP75-EVs or GRP75-EVs with si-UCP1. Primary adipocytes treated with NC-EVs are shown as a negative control. Left: plot of the time course OCR normalized to the protein concentration. Right: calculated respiration levels of basal and proton leaked respiration. g Quantitation of intracellular TG contents in adipocytes treated as described in f . h Immunoblots of GRP75 and UCP1 in differentiated 3T3-L1 adipocytes treated as described in f . i Schematic diagram of in vivo subcutaneous injection of PBS, KYSE150 cells with stable GRP75 overexpression (LV-Flag-GRP75) or negative lentiviral vectors (LV-Control) in six-week-old BALB/c-nude mice ( n = 6 per group). j NBWs of the PBS, LV-Control and LV-Flag-GRP75 groups. k Ratios of iWAT, eWAT, and iBAT to NBW in the three groups. l Representative H&E-stained images of iWAT from the three groups. Scale bar, 50 μm. Quantified adipocyte sizes are shown on the right side. m , n Oxygen consumption volume and heat production of the LV-Control and LV-Flag-GRP75 groups in the metabolic cage housed at 22°C ( n = 6 per group). o Rectal temperatures of the PBS, LV-Control and LV-Flag-GRP75 groups. p Representative IHC images of UCP1 and GRP75 staining in iWAT from the three groups. Scale bar, 50 μm. The magnified image labelled with a red rectangle is shown on the upper right side. Scale bar, 25 μm. q Immunoblotting of GRP75 and UCP1 protein in iWAT from three groups ( n = 4; representative of four biological replicates per group). The data are presented as the mean ± SEM. The exact P values were tested with one-way ANOVA in ( j – l , o ) and unpaired two-tailed Student’s t test in ( m and n )

Article Snippet: The anti-mouse GRP75 (sc-133137) and anti-UCP1 (sc-293418) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Staining, Expressing, Western Blot, Derivative Assay, Negative Control, Protein Concentration, Quantitation Assay, In Vivo, Injection, Over Expression, Control, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: GRP75 binds to and stabilizes ANT2 by decreasing its ubiquitination level. a Identification of endogenous GRP75-interacting proteins. Extracts from the iWAT of YES2 tumour-bearing mice were incubated with protein A/G magnetic beads conjugated with an anti-GRP75 antibody. The eluted proteins were resolved by SDS‒PAGE and visualized by silver staining. The differential gel piece framed by the red rectangle was analysed by mass spectrometry. b Coimmunoprecipitation of endogenous ANT2 and GRP75 in the iWAT of YES2 tumour-bearing mice. Anti-IgG was used as a negative control. c Coimmunoprecipitation of GRP75 and ANT2 in differentiated 3T3-L1 adipocytes transfected with Myc-GRP75 plasmids. d PLA signals and quantification of the combination of anti-GRP75 and anti-ANT2 in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-overexpressing EVs. DAPI, nuclei. Scale bar, 10 μm. e Immunoblotting with anti-GST antibodies was used to detect the binding of recombinant His-ANT2 to GST-GRP75. f Coimmunoprecipitation of ANT2 and truncated domains of GRP75 (bottom panel). Flag-tagged wild-type GRP75 (GRP75-FL) or truncated GRP75 (GRP75-N and GRP75-C) were transfected into HEK293T cells. g Immunoblots (left panel) and real-time qPCR analysis (right panel) of ANT2 and GRP75 in differentiated 3T3-L1 adipocytes transfected with control vector (NC) or Myc-GRP75 plasmids. h Evaluation of the ANT2 half-life in GRP75-overexpressing adipocytes treated with cycloheximide. The indicated proteins were analysed by immunoblotting, and the quantification of ANT2 protein relative to the control β-actin by ImageJ software is shown at the bottom. i Ubiquitination assays of exogenous ANT2 in lysates from differentiated 3T3-L1 adipocytes transfected with NC or GRP75 plasmids. The data are presented as the mean ± SEM. The exact P values were tested with an unpaired two-tailed Student’s t test in ( g )

Article Snippet: The anti-mouse GRP75 (sc-133137) and anti-UCP1 (sc-293418) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Incubation, Magnetic Beads, Silver Staining, Mass Spectrometry, Negative Control, Transfection, Western Blot, Binding Assay, Recombinant, Control, Plasmid Preparation, Software, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: ANT2–GRP75 promotes adipocyte browning by enhancing the interaction with UCP1 and its stability. a , b Quantitation of intracellular TG ( a ) and ATP ( b ) levels in primary adipocytes transfected with control vector (NC) or Flag-ANT2 plasmids (pANT2). c Immunoblots showing ANT2 and UCP1 expression in differentiated 3T3-L1 adipocytes treated as described in a . d , e Quantitation of intracellular TG ( d ) and ATP ( e ) levels in primary adipocytes treated with NC-EVs or GRP75-EVs and transfected with si-NC or si-ANT2. f Immunoblots showing GRP75, ANT2, and UCP1 expression in differentiated 3T3-L1 adipocytes treated as described in d . g Proximity ligation assay signals and enlarged images of the combination of anti-ANT2 and anti-UCP1 in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-overexpressing EVs. DAPI, nuclei. Scale bar, 10 μm. h Immunoblots of UCP1 in differentiated 3T3-L1 adipocytes transfected with NC or Flag-ANT2 plasmids in the presence of cycloheximide. i Ubiquitination assays of UCP1 in lysates from differentiated 3T3-L1 adipocytes transfected with NC or Flag-ANT2 plasmids. j Coimmunoprecipitation of GRP75, ANT2, and UCP1. HEK293T cells were co-transfected with Myc-GRP75, ANT2, and Flag-UCP1. Protein extracts were immunoprecipitated with antibodies against Myc-tag or Flag-tag, followed by immunoblotting with the indicated antibodies. k Exogenous GRP75 enhances the ANT2–UCP1 interaction. Coimmunoprecipitation assays were performed against ANT2 using HEK293T cell lysates with or without transfection of Myc-GRP75. The quantification of protein relative to the control protein β-actin by ImageJ are shown at the bottom in ( c , f and h ). The data are presented as the mean ± SEM. The exact P values were tested with unpaired two-tailed Student’s t tests in ( a and b ) and one-way ANOVA in ( d and e )

Article Snippet: The anti-mouse GRP75 (sc-133137) and anti-UCP1 (sc-293418) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Quantitation Assay, Transfection, Control, Plasmid Preparation, Western Blot, Expressing, Proximity Ligation Assay, Ubiquitin Proteomics, Immunoprecipitation, FLAG-tag, Two Tailed Test

Journal: Signal Transduction and Targeted Therapy

Article Title: GRP75 triggers white adipose tissue browning to promote cancer-associated cachexia

doi: 10.1038/s41392-024-01950-w

Figure Lengend Snippet: GRP75 inhibitors alleviate adipocyte browning in vitro and in vivo. a Quantitation of intracellular TG content in differentiated 3T3-L1 adipocytes treated with NC-EVs or GRP75-EVs in the presence or absence of the GRP75 inhibitor withanone (WNN). b OCR of differentiated adipocytes as described in a . Left: plot of the time course OCR normalized to the protein concentration. Right: calculated respiration levels of basal and proton-leaked OCRs. c Immunoblots of GRP75, ANT2, and UCP1 in differentiated adipocytes, as shown in a . d Coimmunoprecipitation of GRP75 and ANT2 in HEK293T cells treated with or without WNN. HEK293T cells were transfected with GFP-GRP75 plasmids and Flag-ANT2 plasmids. e Experimental design of the in vivo experiment. YES2 tumour-bearing mice were intraperitoneally injected with DMSO (DMSO), WNN (5 mg/kg), cisplatin (10 mg/kg, CDDP), or a combination of WNN with cisplatin (Combo). The PBS and PF groups included tumour-free mice injected with vehicle. f NBWs of the six groups described in e on day 48. g Ratios of iWAT (left) and eWAT (right) to NBW in e on day 48. h Representative IHC images of UCP1 in the iWAT from six groups. Scale bar, 50 μm. Magnified images labelled with a red rectangle are shown in the bottom panel. Scale bar, 25 μm. i – j Immunoblots of GRP75, ANT2, and UCP1 in the iWAT of the DMSO and WNN groups ( i ) or the CDDP and Combo groups ( j ) ( n = 6; representative of six biological replicates per group). k Working model for how tumour extracellular vesicular GRP75 regulates white adipocyte browning in cachexia progression (created by Biorender.com). The data are presented as the mean ± SEM. n = 6 (PBS and PF), n = 8 (DMSO), n = 12 (WNN), n = 10 (CDDP) and n = 9 (Combo). The exact P values were tested with one-way ANOVA in ( a , b , f , and g )

Article Snippet: The anti-mouse GRP75 (sc-133137) and anti-UCP1 (sc-293418) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: In Vitro, In Vivo, Quantitation Assay, Protein Concentration, Western Blot, Transfection, Injection

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet: Chronic administration of AOPP induced PC defects in intestinal crypts of mice (A and B) Representative images of HE staining for PC number quantification and immunohistochemical staining for Lysozyme in intestine sections of mice with or without AOPP treatment. These images were representative of results from each experimental group, each with five mice. Images showed that AOPP challenge reduced the PC number and lysozyme expression in intestinal crypts compared with vehicle-treated mice. Scale bar, 20 μm. (C) Representative Western blotting and densitometric quantification showed the protein level of Lysozyme in isolated intestinal crypts of mice with or without AOPP administration. (D and E) Bar graphs representing qPCR analysis showed the mRNA levels of Lysozyme and cryptdins in isolated intestinal crypts of mice with or without AOPP administration. Relative protein and mRNA levels were normalized by GAPDH, respectively. Data were represented by median with range, n = 5 mice; error bars indicating range. Student’s t test, ∗∗p < 0.01 versus vehicles. Vehicle, PBS treatment for 28 days; AOPP, AOPP treatment for 28 days; qPCR, quantitative real-time polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: Anti-mouse GAPDH, anti-rabbit GRP75, anti-rabbit VDAC1, anti-rabbit Mfn1/2 and anti-rabbit PDI were all purchased from Proteintech (Wuhan, China).

Techniques: Staining, Immunohistochemical staining, Expressing, Western Blot, Isolation, Real-time Polymerase Chain Reaction

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet: Chronic challenge of AOPP induced ER stress in intestinal crypts of mice (A and B) Representative images of immunohistochemical staining for GRP78 and CHOP in mice with or without AOPP challenge. These images were representative of results from each experimental group, each with five mice. Images showed that AOPP treatment elevated GRP78 and CHOP levels in intestinal crypts compared with vehicle-treated mice. Scale bar, 20 μm. (C) Representative Western blotting and densitometric quantification showed the protein levels of GRP78, CHOP and IRE1α in isolated intestinal crypts of mice with or without AOPP challenge. (D and E) Bar graphs representing qPCR analysis showed the levels of GRP78 and CHOP mRNA in isolated intestinal crypts of mice with or without AOPP challenge. Relative protein and mRNA levels were normalized by GAPDH, respectively. Data were represented by median with range, n = 5 mice; error bars indicating range. Student’s t test, ∗p < 0.05 and ∗∗p < 0.01 versus vehicles.

Article Snippet: Anti-mouse GAPDH, anti-rabbit GRP75, anti-rabbit VDAC1, anti-rabbit Mfn1/2 and anti-rabbit PDI were all purchased from Proteintech (Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Western Blot, Isolation

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet: AOPP-mediated PC defects depended on ER stress in intestinal crypts of mice (A and B) Representative images of HE staining for PC number quantification and immunohistochemical staining for Lysozyme in intestine sections of mice that underwent intraperitoneal administration of AOPP with or without TUDCA. These images were representative of results from each experimental group, each with five mice. Images reveled that treatment with TUDCA alleviated AOPP-mediated decreasing PC number and lysozyme expression in intestinal crypts compared with AOPP-treated mice. Scale bar, 20 μm. (C) Representative Western blotting and densitometric quantification revealed the protein level of Lysozyme in isolated intestinal crypts of mice that underwent intraperitoneal administration of AOPP with or without TUDCA. (D and E) Bar graphs representing qPCR analysis revealed the levels of mRNA of Lysozyme and cryptdins in isolated intestinal crypts of mice that underwent intraperitoneal administration of AOPP with or without TUDCA. Relative protein and mRNA levels were normalized by GAPDH, respectively. Data were represented by median with range, n = 5 mice; error bars indicating range. ANOVA, ∗∗p < 0.01 versus vehicles; # p < 0.05 and ## p < 0.01 versus AOPP-treated mice.

Article Snippet: Anti-mouse GAPDH, anti-rabbit GRP75, anti-rabbit VDAC1, anti-rabbit Mfn1/2 and anti-rabbit PDI were all purchased from Proteintech (Wuhan, China).

Techniques: Staining, Immunohistochemical staining, Expressing, Western Blot, Isolation

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet: TUDCA inhibited AOPP-resulted ER stress in intestinal crypts of mice (A and B) Representative images of immunohistochemical staining for GRP78 and CHOP in mice that underwent intraperitoneal administration of AOPP with or without TUDCA. These images were representative of results from each experimental group, each with five mice. Images revealed that treatment with TUDCA attenuated AOPP-induced increased GRP78 and CHOP levels in intestinal crypts compared with AOPP-treated mice. Scale bar, 20 μm. (C) Representative Western blotting and densitometric quantification revealed the protein levels of GRP78, CHOP and IRE1α in isolated intestinal crypts of mice that underwent intraperitoneal administration of AOPP with or without TUDCA. (D and E) Bar graphs representing qPCR analysis revealed the levels of GRP78 and CHOP mRNA in isolated intestinal crypts of mice that underwent intraperitoneal administration of AOPP with or without TUDCA. Relative protein and mRNA levels were normalized by GAPDH, respectively. Data were represented by median with range, n = 5 mice; error bars indicating range. ANOVA, ∗∗p < 0.01 versus vehicle; # p < 0.05 and ## p < 0.01 versus AOPP-treated mice. AOPP+TUDCA, AOPP treatment plus daily intraperitoneal injection of TUDCA; TUDCA, tauroursodeoxycholic acid, an inhibitor of ER stress.

Article Snippet: Anti-mouse GAPDH, anti-rabbit GRP75, anti-rabbit VDAC1, anti-rabbit Mfn1/2 and anti-rabbit PDI were all purchased from Proteintech (Wuhan, China).

Techniques: Immunohistochemical staining, Staining, Western Blot, Isolation, Injection

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet:

Article Snippet: Anti-mouse GAPDH, anti-rabbit GRP75, anti-rabbit VDAC1, anti-rabbit Mfn1/2 and anti-rabbit PDI were all purchased from Proteintech (Wuhan, China).

Techniques: Recombinant, Lysis, Protein Quantitation, Isolation, ATP Assay, Software

Journal: iScience

Article Title: Advanced oxidation protein products induce Paneth cells defects by endoplasmic reticulum stress in Crohn's disease

doi: 10.1016/j.isci.2023.107312

Figure Lengend Snippet: Primer sequences

Article Snippet: Anti-mouse GAPDH, anti-rabbit GRP75, anti-rabbit VDAC1, anti-rabbit Mfn1/2 and anti-rabbit PDI were all purchased from Proteintech (Wuhan, China).

Techniques:

Journal: Nature communications

Article Title: Enhanced Ca 2+ -channeling complex formation at the ER-mitochondria interface underlies the pathogenesis of alcohol-associated liver disease.

doi: 10.1038/s41467-023-37214-4

Figure Lengend Snippet: Fig. 8 | Graphical illustration of PDK4-mediated Ca2+-channeling complex for- mation at the ER-mitochondria contact site promotes mitochondrial dys- function in alcohol-associated liver disease. Ca2+-channeling complex formation in MAM is augmented in alcohol-associated liver disease (ALD). An increase in PDK4 expression enhances the MAM Ca2+-channeling complex formation via the phos- phorylation of GRP75 to promote mitochondrial Ca2+ accumulation and dysfunc- tion in ALD. ROS: Reactive Oxygen Species, OCR: Oxygen Consumption Rate, MMP: Mitochondrial Membrane Potential.

Article Snippet: Primary antibodies used in in situ PLA experiments were anti-rabbit VDAC1 (Abcam, ab15895; 1:100 dilution), anti-mouse monoclonal IP3R1 (Santa Cruz; sc-271197; Clone E8; 1:50 dilution), anti-mouse monoclonal GRP75 (Santa Cruz, sc133137; Clone D9; 1:50 dilution), anti-rabbit IP3R1 (Invitrogen, PA1-901; 1:100 dilution), anti-rabbit FLAG (Cell signaling, #2368; 1:100 dilution) and antimouse HA (Santa Cruz, sc7392; Clone F-7; 1:50 dilution).

Techniques: Expressing, Membrane

Journal: Cell death & disease

Article Title: Ultrastructural and proteomic profiling of mitochondria-associated endoplasmic reticulum membranes reveal aging signatures in striated muscle.

doi: 10.1038/s41419-022-04746-4

Figure Lengend Snippet: Fig. 3 Mitochondria-associated membranes (MAMs) purified and identified from rat heart and GA muscle. A Scheme for mitochondria- associated membranes (MAMs) using Percoll gradient centrifugation. B Subcellular fractions prepared from rat heart were validated by transmission electronic microscopy (TEM). Bar = 0.5 μm. C Protein components of subcellular fractions prepared from rat heart and GA muscle were revealed by immunoblot analysis. CALR (calreticulin) and GAPDH were used as markers for ER and cytosolic fractions, respectively; IP3R and CANX (calnexin) was considered as markers of both ER and MAMs, whereas VDAC1 and GRP75 was adopted as markers of both mitochondria and MAMs. D and E The protein content (in mg) of MAMs, pure mitochondria and microsome was quantified and normalized by total tissue mass (in g) to obtain the relative protein abundance of subcellular fractions in heart (D) and GA muscle (E) (n = 6 rats per group, data represent mean ± SD. Variance was similar between groups being compared. *P < 0.05; **P < 0.01). PNS post-nuclear supernatant, Mc crude mitochondria, Mp pure mitochondria, Microsome microsome/ER fraction, MAM mitochondria-associated membrane, Cyto cytosol.

Article Snippet: After blocking in 5% non-fat milk, the following primary antibodies were used: anti-Calnexin rabbit pAb (1:1000, Cat.A15631, ABclonal, Wuhan, China), anti-Calreticulin rabbit pAb (1:1000, Cat.A18013, ABclonal), anti-GRP75 mouse mAb (1:1000, Cat. sc-133137, Santa Cruz, CA, USA), anti-IP3R rabbit pAb (1:1000, Cat. ab5804, Abcam, MA, USA), antiVDAC1 mouse mAb (1:1000, Cat. Ab186321, Abcam), anti-TOMM20 rabbit mAb (1:1000, Cat. Ab187735, Abcam,), anti-SAMM50 rabbit pAb (1:1000, Cat. ab246987, Abcam), anti-MIC60 (1:1000, Cat. 10179-1-AP, Proteintech, Chicago, USA), anti-MTX1 rabbit pAb (1:1000, ab233205, Abcam), anti- Cell Death and Disease (2022) 13:296 GAPDH Rabbit mAb (1:5000, Cat. A19056, ABclonal).

Techniques: Gradient Centrifugation, Transmission Assay, Microscopy, Western Blot, Quantitative Proteomics, Membrane