goat anti cd36 antibody  (R&D Systems)

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, Control, 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, Control, 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: Phospho-proteomics, 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, Phospho-proteomics, Binding Assay