Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: High CD36 expression in S508A mutant and effect of CD36 RNAi. A – F , staining of CD36 ( green ) in HepG2 cell lines. G , quantitation of CD36 staining for the six cell lines (see ). H – J , S508A mutants stained with CD36 ( green ), F-actin ( red ), and DAPI ( blue ). CD36 and BC expression of untreated ( H ) and RNAi control-treated ( I ) S508A mutant cells versus RNAi to CD36-treated S508A mutant cells ( J ). BCs are shown with arrows . K – N , lipid droplet staining ( green ) of S508A mutant cells. Control RNAi treatment at 20× ( K ) and 40× ( L ). RNAi to CD36 treatment at 20× ( M ) and 40× ( N ). O , mRNA expression levels in cell lines by qRT-PCR (in triplicate, relative to GAPDH). P , treatment of cell lines with no RNAi, control RNAi, RNAi-1, RNAi-2, and RNAi-1 plus RNAi-2 to CD36 (in triplicate, relative to GAPDH). Q , cell surface levels of CD36 on four cell lines. R , SDS gel analysis of CD36 expression in six cell lines. KO, knockout; SA, S508A mutant; SD, S508D mutant; WT, wild type.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Expressing, Mutagenesis, Staining, Quantitation Assay, Quantitative RT-PCR, SDS-Gel, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Nuclear expression of LKB1 in WT, CEACAM1 −/− , and S508A and S508D mutants and AMPK expression. LKB1 nuclear expression in WT ( A ) and in CEACAM1 −/− ( B ) and in S508A ( C ) and S508D ( D ) mutants ( red = LKB1, blue = DAPI). Percent LKB1 positive nuclei per 50 cells counted for three fields ±SEM shown underneath each panel. Magnification 20×. E , triple staining of S508A mutant for CD36 ( green ), LKB1 ( red ) and nuclei ( blue ). Magnification 40×. F , immunoblots for the detection of activated AMPK ( green channel ) in WT, CEACAM1 KO, and CEACAM1 mutants (tubulin in red channel ). Twenty micrograms of protein lysate loaded per lane.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Expressing, Staining, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: PCSK9 expression affects CD36 expression in the S508A mutant. PCSK9 expression in WT ( A ) and in CEACAM1 S508A mutant ( B ) HepG2 cells. Triple stained for PCSK9 ( green ), F-actin ( red ), and nuclei ( blue ). Effect of control RNAi ( C ) and PCSK9 RNAi ( D ) on CD36 expression ( green ) in CEACAM1 S508A mutant HepG2 cells. Lack of an effect of control RNAi ( E ) and CD36 RNAi ( F ) on PCSK9 expression ( green ). G , effect of PCSK9 RNAi on PCSK9 mRNA expression as measured by qRT-PCR. H , effect of PCSK9 RNAi on CD36 mRNA expression as measured by qRT-PCR in triplicate.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Expressing, Mutagenesis, Staining, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Tyrosine phosphorylation of CEACAM1 by insulin versus Src and coexpression of SRC and CD36 with CEACAM1 in mutant S508A cells. A , lysates (20 μg) from WT cells treated before and after with insulin (15 μg/ml) over time were IPed with anti-CEACAM1 antibody and immunoblotted for phosphotyrosine. B , lysates (20 μg) from cells treated before and after with insulin for 30 min were IPed with anti-CEACAM1 antibody and immunoblotted for phosphotyrosine (normalized for CEACAM1 signal in a parallel immunoblot). C and D , equal amounts of lysates from S508A ( C ) or S508D ( D ) mutants treated with insulin or inhibitors of Src (BOS, 10 μM), PI3K (LY294002, 20 μM), or CaMK2 (KN93, 20 μM), IPed with anti-CEACAM1 antibody and run on SDS gels were blotted for phosphotyrosine. Mutant S508A cells were stained for Src ( red ), CEACAM1 ( green ), DAPI ( blue ), and overlayed ( E ) or for Src ( red ), CD36 ( green ), DAPI ( blue ), and overlayed ( F ). Arrows indicate examples of prominent BCs with yellow staining indicating overlap of Src with CEACAM1 or CD36. Magnifications were 40× ( E ) and 20× ( F ).

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Mutagenesis, Western Blot, Staining

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Coimmunoprecipitation of CEACAM1, Src, LB1, and Annexin A2 with CD36. CD36 was immunoprecipitated from Ser508A mutant HepG2 cells, pretreated or not with insulin, run on SDS gels, and immunoblotted for CD36, CEACAM1, Src, LKB1, and Annexin A2 (AnxA2). Equal amounts of protein were probed on immunoblots.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Immunoprecipitation, Mutagenesis, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human CEACAM1-LF regulates lipid storage in HepG2 cells via fatty acid transporter CD36

doi: 10.1016/j.jbc.2021.101311

Figure Lengend Snippet: Model of FA import by CD36 and lipid droplet management in WT, CEACAM1 −/− , and CEACAM1 mutants in HepG2 cells. A , lipid droplets (LDs) increase in CEACAM1 −/− versus WT cells and are reduced in S508A mutant cells along with an increase in size of bile canaliculi (BCs). B , CD36 is found in a complex with CEACAM1, Src, LKB1, and AMPK. Src can phosphorylate CEACAM1 on Y493 and Y520 located on either side of S508. LKB1 can phosphorylate AMPK; however, phosphorylated LKB1 can translocate to the nucleus, reducing phosphorylation of AMPK. CD36 can be internalized to endosomes after binding long chain fatty acids and shunted to either lysosomes for degradation or to BCs. C , phosphorylation of S512 by PKA followed by phosphorylation of S508 by GSK3β can lead to high lipid accumulation. Src phosphorylation of Y493 leads to high lipid accumulation, while phosphorylation of Y520 leads to low lipid accumulation. Phosphorylation of both tyrosines is influenced by the phosphorylation status of S508. Thus, the null mutants of Y493 and Y520 can abrogate their normal lipid regulation functions.

Article Snippet: Goat anti-human CD36 (50 μg; R&D, AF1955) was immobilized on a Thermo Scientific IP column, 26149, washed 3× with lysis buffer, incubated with cell lysates, washed 3× with lysate buffer, and eluted with 2× SDS sample buffer.

Techniques: Mutagenesis, Binding Assay

Journal: JCI Insight

Article Title: Whole-body deletion of Endospanin 1 protects from obesity-associated deleterious metabolic alterations

doi: 10.1172/jci.insight.168418

Figure Lengend Snippet: ( A ) Detection of Flag-CD36 in 6Myc-Endo1 immunoprecipitate from HEK293T cells coexpressing 6Myc-Endo1 and Flag-CD36 compared with HEK293T cells transfected with Flag-CD36 alone. Representative blots of 3 independent experiments. ( B ) Detection of 6Myc-Endo1 after Flag-CD36 immunoprecipitation from HEK293T cells coexpressing 6Myc-Endo1 and Flag-CD36 compared with HEK293T cells transfected with 6Myc-Endo1 alone. Representative blots of 3 independent experiments. ( C ) Kinetics of Endo1 and CD36 protein expression in differentiated adipocytes during differentiation of adipocyte precursors isolated from the stromal vascular fraction of the s.c. adipose tissue of WT mice into mature white adipocytes. Results are expressed as mean ± SEM of 8 independent experiments. One representative Western blot is shown. ( D ) Confocal immunofluorescence detection and colocalization of Endo1 (rabbit anti-Endo1) and CD36 (goat anti-CD36) in differentiated adipocytes. Nuclei (blue) are stained with fluorescent DAPI dye. Scale bar: 20 μm. Representative images of 4 independent experiments. ( E ) Detection of endogenous Endo1 and CD36 after immunoprecipitation with Endo1 or CD36 antibodies from lysates of visceral adipose tissue (VAT), gonadal adipose tissue (GAT), and s.c. adipose tissue (SAT) of WT and KO mice. Endo1-KO adipocytes and tissues were used as negative controls. The molecular weights of protein markers are indicated (kDa). Representative blots of 2 independent experiments.

Article Snippet: Next, cells were incubated with primary Endo1 (1:500) (generated as described previously; ref. ) and primary CD36 (1:1,000) (RD Systems, catalog AF2519), primary Endo1 (1:500) and primary 53K (1:1,000, Abcam, catalog ab27043), or primary GM130 (1:1,000, Abcam, catalog ab169276) antibodies overnight at 4°C.

Techniques: Transfection, Immunoprecipitation, Expressing, Isolation, Western Blot, Immunofluorescence, Staining

Journal: JCI Insight

Article Title: Whole-body deletion of Endospanin 1 protects from obesity-associated deleterious metabolic alterations

doi: 10.1172/jci.insight.168418

Figure Lengend Snippet: ( A ) Cell surface expression of CD36 in mature white adipocytes. ** P < 0.01 versus WT. Results are expressed as mean ± SEM ( n = 4). Two-tailed t test. ( B ) Total CD36 expression in gonadal adipose tissue (GAT) and s.c. adipose tissue (SAT) . The molecular weights of protein markers are indicated (kDa). Results are expressed as mean ± SEM ( n = 5). ( C ) Immunofluorescence images of CD36 cell surface expression in differentiated white adipocytes (left panel). Level of cellular fluorescence determined by corrected total cell fluorescence per area (CTCF/area). Results are expressed as mean ± SEM ( n = 9). *** P < 0.005 versus WT. Two-tailed t test (right panel). Scale bar: 20 μm. ( D ) Lipid uptake in differentiated adipocytes, mature adipocytes, and differentiated myotubes. Results are expressed as mean ± SEM ( n = 5–6). * P < 0.05; ** P < 0.01; **** P < 0.001 versus basal. † P < 0.05; †† P < 0.01; †††† P < 0.001 versus WT. One-way ANOVA with Bonferroni correction.

Article Snippet: Next, cells were incubated with primary Endo1 (1:500) (generated as described previously; ref. ) and primary CD36 (1:1,000) (RD Systems, catalog AF2519), primary Endo1 (1:500) and primary 53K (1:1,000, Abcam, catalog ab27043), or primary GM130 (1:1,000, Abcam, catalog ab169276) antibodies overnight at 4°C.

Techniques: Expressing, Two Tailed Test, Immunofluorescence, Fluorescence

Journal: bioRxiv

Article Title: Small proline-rich proteins (SPRRs) are epidermally produced antimicrobial proteins that defend the cutaneous barrier by direct bacterial membrane disruption

doi: 10.1101/2021.09.01.458578

Figure Lengend Snippet: (A-D) qRT-PCR analysis of Sprr1a and Sprr2a gene expression in mouse dorsal skin tissue. (A) Germ-free mice (GF) compared to conventionally raised mice (CV). (B) PBS treated mouse skin compared to LPS intradermal injection mouse skin in WT or MYD88 −/− mouse. (C) Mouse intradermally-injected by vehicle (PBS), E.coli or heat-inactivated (HI) E.coli. (D) Untreated mouse skin compared to wounded skin abraded in a crosshatch pattern by a 15-blade scalpel. Means ± SEM are plotted. *P < 0.05; **P < 0.01; ****P < 0.0001ns, not significant by unpaired t test. (E) Immunofluorescence staining of SPRR1A expression in mice skin. CD36 was used as marker of sebocyte cells. Nuclei are stained with DAPI (Blue). Epidermis and sebaceous gland indicated with an arrow. White dashed line separates the epidermis and dermis. Yellow dashed line indicates the outline of SG. Scale bar, 50 μm.

Article Snippet: Slides were blocked with 10% FBS, 1% BSA and 1% Triton X-100 in PBS, and then incubated with primary antibodies against mouse SPRR1A (Thermo Fisher PA5-26062), mouse CD36 (R&D AF2519), rabbit IgG isotype control (Thermo Fisher 02-6102) or goat IgG isotype control (Thermo Fisher 02-6202) using 1:100 dilutions at 4°C overnight.

Techniques: Quantitative RT-PCR, Expressing, Injection, Immunofluorescence, Staining, Marker

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Expression of CD36 in preadipocytes is induced in adipose tissue of obese patients. General health survey data were collected from 28,035 patients who underwent health examinations at the Affiliated Hospital of CQMU. Body mass index (BMI), waistline and serum hs-CRP measurements were divided into quartiles. The ranges of the different quartiles of BMI were <21.55 kg/m 2 (1st quartile), 21.55–23.66 kg/m 2 (2nd quartile), 23.66-25.81 kg/m 2 (3rd quartile) and >25.81 kg/m 2 (4th quartile). The ranges of the different quartiles of waist circumference were <76 cm (1st quartile), 76–82 cm (2nd quartile), 82–88 cm (3rd quartile) and 88 cm (4th quartile). The ranges of the different quartiles of hs-CRP were 0-0.30 mg/L (1st quartile), 0.31–0.60 mg/L (2nd quartile), 0.61–1.25 mg/L (3rd quartile) and >1.25 mg/L (4th quartile). Human visceral adipose tissue samples were obtained from patients who underwent laparoscopic cholecystectomy. The subjects were subdivided into two groups: non-obese patients (18.5 kg/m 2

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Expressing, Staining, Immunohistochemical staining, Double Immunofluorescence Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Expression of CD36 in preadipocytes is induced in HFD-fed mice accompanied with lysosomal impairment. C57BL/6J mice were fed a HFD (5 males and 5 females) or normal diet (ND) (5 males and 5 females) for 14 weeks. (a) Double immunofluorescence staining for CD36 and Pref1 in sections of mouse adipose tissue. The arrow indicates the colocalization area. (b) Preadipocytes (CD146 − /CD34 + cells) in mouse adipose tissue were sorted by a FACScan flow cytometer. CD36 expression (c) and lysosome function (d-e) in CD146 − /CD34 + cells were detected by a FACScan flow cytomete. * p <0.05 compared with the C57-ND group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Expressing, Double Immunofluorescence Staining, Flow Cytometry

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: FFA upregulates CD36 expression and induces lysosomal impairment, lipid accumulation and inflammation in 3T3L1 preadipocytes. (a) Cell viability assay in 3T3L1 preadipocytes treated with FFA for 60 h. (b) The relative protein expression of CD36 in 3T3L1 preadipocytes after treatment with FFA (0.4 mM PA+0.2 mM OA) for 15 h, 30 h and 60 h was detected by western blotting ( n= 3). * p <0.05 compared with 0h. (c) CD36 expression in plasma membrane (PM) of 3T3L1 preadipocytes treated with FFA for 0h and 60 h was detected by a FACScan flow cytometer ( n= 3). (d) Lysosome function in 3T3L1 preadipocytes treated with FFA for 15 h and 60 h were detected by a FACScan flow cytometer ( n= 3). (e) BODIPY staining of 3T3L1 preadipocytes treated with or without FFA (FFA group or control (CTL) group) for 60 h ( n= 3). (f) Relative mRNA levels of MCP1, TNF α, IL-6 and IL-1β in 3T3L1 preadipocytes treated with FFA for 60 h ( n= 3). * p <0.05 compared with the CTL group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Expressing, Viability Assay, Western Blot, Membrane, Flow Cytometry, Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Forced CD36 upregulation induces lipid accumulation and inflammation in 3T3L1 preadipocytes were infected with a recombinant lentivirus containing CD36 cDNA (wtCD36 OE preadipocytes) or empty vector (NC preadipocytes) and these cells were selected with puromycin. (a) Western blot analysis of CD36, NF-κB p65, LaminB1, caspase-1, and IL-1β in NC and wtCD36 OE preadipocytes ( n= 3). (b) The quantitative data of protein expression in A. (c) Relative mRNA levels of MCP1, TNF α, IL-6 and IL-1β in NC and wtCD36 OE preadipocytes ( n= 3). (d) Migration assay. A migration assay was performed by using Transwell migration chambers (8-μm pore size) to assess THP-1 cell migration co-cultured with the 3T3L1 preadipocyte lines ( n= 3). (e) BODIPY staining and oil red O staining of NC and wtCD36 OE preadipocytes ( n= 3). * p< 0.05 compared with the NC group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Infection, Recombinant, Plasmid Preparation, Western Blot, Expressing, Migration, Pore Size, Cell Culture, Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Forced CD36 upregulation impairs lysosomal function and lipophagy in 3T3L1 preadipocytes. (a) Western blot analysis of P62 in NC and wtCD36 OE preadipocytes ( n= 3). (b) Double fluorescent staining of mRFP-GFP-LC3 fusion protein in NC and wtCD36 OE preadipocytes. (c) Lysosome function in NC and wtCD36 OE preadipocytes were detected by a FACScan flow cytometer ( n= 3). (d) Representative images showed double immunofluorescence staining for BODIPY and Lysotracker in wtCD36 OE and NC preadipocytes. The arrow indicates the colocalization area. * p< 0.05 compared with the NC group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Western Blot, Staining, Flow Cytometry, Double Immunofluorescence Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: Forced CD36 upregulation promotes IP3R1-mediated Ca 2+ transport from the ER to lysosomes in preadipocytes. (a) Representative images showed double immunofluorescence staining for OG-BAPTA-5N and lysotracker in wtCD36 OE and NC preadipocytes. (b) Western blot analysis of IP3R1 and P-IP3R1 (Tyr353) in wtCD36 OE preadipocytes and NC preadipocytes ( n= 3). After wtCD36 OE preadipocytes were treated with or without 2APB (0.5 μM) for 1 h, lysosome function (c-d) were detected by a FACScan flow cytometer ( n= 3). (e) BODIPY staining ( n= 3). (f) Mitochondrial reactive oxygen species assay ( n= 3). (g) Relative mRNA levels of MCP1, TNF α, IL-6 and IL-1β. * p< 0.05 compared with the NC group ( n= 3). # p< 0.05 compared with the wtCD36 OE group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Double Immunofluorescence Staining, Western Blot, Flow Cytometry, Staining

Journal: EBioMedicine

Article Title: Obesity induces preadipocyte CD36 expression promoting inflammation via the disruption of lysosomal calcium homeostasis and lysosome function

doi: 10.1016/j.ebiom.2020.102797

Figure Lengend Snippet: CD36 coordinates with Fyn to phosphorylate IP3R1, inducing lysosomal Ca 2+ overload and inflammation in preadipocytes. We constructed a lentivirus containing CD36 with a palmitoylation site mutation and infected the 3T3L1 cells with lentivirus (mt∆CD36 OE preadipocytes). (a) Co-IP for CD36 and Fyn in wtCD36 OE preadipocytes and mt∆CD36 OE preadipocytes ( n= 3). (b) Western blot analysis of IP3R1 and P-IP3R1 (Tyr353) in wtCD36 OE preadipocytes and mt∆CD36 OE preadipocytes ( n= 3). (c) Cells were incubated for 30 min in the presence of 0.5 μM fluo-4 AM, a Ca 2+ indicator dye, to detect total intracellular Ca 2+ ( n= 3). (d) ER Ca 2+ release assay. Cells were incubated with fluo-4 AM (0.5 μM) for 30 min. Intracellular Ca 2+ was monitored prior to and following exposure to 1 μM thapsigargin ( n= 3). (e) Representative images showed double immunofluorescence staining for OG-BAPTA-5N and Lysotracker in NC preadipocytes, wtCD36 OE preadipocytes (treated with or without PP2) and mt∆CD36 OE preadipocytes. (f) Western blot analysis of NF-kB and IL-1β in wtCD36 OE preadipocytes and mt∆CD36 OE preadipocytes ( n= 3). * p< 0.05 compared with the NC group. # p< 0.05 compared with the wtCD36 OE group.

Article Snippet: Paraffin-embedded visceral adipose tissue sections were incubated with anti-CD36 (Novus Cat# NB400-144, RRID:AB_10003498) anti-mouse Pref1 (R&D Systems, AF8277) or anti-human Pref1 (R&D Systems, MAB1144-SP), and then incubated with FITC-conjugated anti-rabbit IgG (ZSGB-Bio Cat# ZF-0311, RRID:AB_2571576), TRITC-conjugated anti-goat IgG (ZSGB-Bio, Cat# ZF-0317) or TRITC-conjugated anti-mouse IgG (ZSGB-Bio Cat# ZF-0313, RRID:AB_2571577).

Techniques: Construct, Mutagenesis, Infection, Co-Immunoprecipitation Assay, Western Blot, Incubation, Release Assay, Double Immunofluorescence Staining

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

doi: 10.33594/000000038

Figure Lengend Snippet: Fig. 2. Demonstration of (A) representative blots of TSP1, TSP2, CD36 and GAPDH by immunoblotting at different stages of CL development in the riverine buffalo. The relative molecular weight of each protein is shown along the right side of each blot. The luteal proteins were loaded @ 100 mg/ well and resolved in 10% SDS-PAGE followed by electrotransfer to a PVDF membrane. Protein specific antibodies were used @ 1:500 while secondary antibody @1:2000 dilutions. GAPDH was used as reference protein. Relative expression of TSP and its receptors was analysed by densitometry using image J software (n = 6/group). One-way ANOVA to determine if treatment groups were significantly different. Tukey HSD test was done to find the pair-wise mean differences. Each bar represents Mean ± SEM. Different superscripts denote statistical significance (p<0.05). Abbreviations: CL, Corpus Luteum; TSP, Thrombospondin; CD, Cluster of differentiation.

Article Snippet: Antibodies and Growth factor Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc362257; Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot, Molecular Weight, SDS Page, Electrotransfer, Membrane, Expressing, Software

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Transcriptional Regulation of Thrombospondins and Its Functional Validation through CRISPR/Cas9 Mediated Gene Editing in Corpus Luteum of Water Buffalo (Bubalus Bubalis).

doi: 10.33594/000000038

Figure Lengend Snippet: Fig. 5. Immunohistochemical localization of CD36 in the late CL stage of riverine buffalo. The 5 μm thick sections of CL were deparaffinised and rehydrated, followed by antigen retrieval. Primary TSP1 antibody was used at 1:200 while the FITC was used at 1:500. DAPI was used to counterstain nucleus. Fluorescent signals were captured by microscopy (Carl Zeiss Micro Imaging GmbH). Representative images from (A) bright field, (B) through (C) indicate intense immunoreactivity in late stages of CL which was localized predominantly in the cytoplasm of luteal cells. No primary antibody was used in the negative control (D). Scale bar = 50 μm. Abbreviations: LL, Large luteal cell; SL, Small luteal cell; FITC, Fluorescein isothiocyanate; DAPI, 40, 6-diamidino-2-phenylindole dihydrochloride.

Article Snippet: Antibodies and Growth factor Immunoblotting and immunohistochemistry were performed using goat polyclonal GAPDH (sc-48166; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse monoclonal TSP1 (sc-393504; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal TSP2 (sc-12313; Santa Cruz Biotechnology, Inc., Dallas, TX), goat polyclonal CD36 (sc-5522; Santa Cruz Biotechnology, Inc., Dallas, TX), mouse anti-goat IgG-HRP (sc-2354; Santa Cruz Biotechnology, Inc., Dallas, TX), goat anti-mouse IgG-HRP (sc-2005; Santa Cruz Biotechnology, Inc., Dallas, TX) bovine anti-goat IgG-CFL-647 (sc-362284, Lot#B0312) and goat anti-mouse IgG-CFL-647 (sc362257; Santa Cruz Biotechnology, Inc.).

Techniques: Immunohistochemical staining, Microscopy, Imaging, Negative Control