Anti-Scavenging Receptor SR-BI antibody [EP1556Y]

• FuncS

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Functional Studies - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

THP1 cells were incubated at 37°C for 40h with vehicle control (0 μM) and different concentrations of sodium salicylate (ab120746). Decreased expression of scavenging receptor SR-BI in THP1 cells correlates with an increase in sodium salicylate concentration, as described in literature.

Whole cell lysates were prepared with RIPA buffer (containing protease inhibitors and sodium orthovanadate), 10 μg of each were loaded on the gel and the WB was run under reducing conditions. After transfer the membrane was blocked for an hour using 5% BSA before being incubated with unpurified ab52629 at 1/2000 dilution and ab8227 at 1 μg/ml overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP (ab97051) at 1/10000 dilution and visualised using ECL development solution.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

Immunohistochemical staining of paraffin embedded human liver with purified ab52629 at a working dilution of 1/500. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• WB

Lab

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/1000 dilution

Lane 1:

Human fetal liver lysate at 20 µg

Lane 2:

HepG2 lysate at 20 µg

Lane 3:

PC-3 cell lysate at 20 µg

Secondary

All lanes:

Anti-rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 60 kDa

Observed band size: 80 kDa

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• WB

Lab

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : Scavenging Receptor SR-BI knockout HAP1 whole cell lysate (20 μg)
Lane 3 : HepG2 whole cell lysate (20 μg)
Lane 4 : Human liver whole cell lysate (20 μg)

Lanes 1 - 4 : Merged signal (red and green). Green - ab52629 observed at 80 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab52629 was shown to specifically react with Scavenging Receptor SR-BI in wild-type HAP1 cells as signal was lost in Scavenging Receptor SR-BI knockout cells. Wild-type and Scavenging Receptor SR-BI knockout samples were subjected to SDS-PAGE. ab52629 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629)

Predicted band size: 60 kDa

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• WB

Lab

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

False colour image of Western blot : Anti-Scavenging Receptor SR-BI antibody [EP1556Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab52629 was shown to bind specifically to Scavenging Receptor SR-BI. A band was observed at 70/75 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SCARB1 knockout cell line ab282646 (knockout cell lysate ab283046). To generate this image, wild-type and SCARB1 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SCARB1 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human SCARB1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-scarb1-knockout-hek-293t-cell-line-ab282646'>ab282646</a>)

Lane 3:

Human Liver cell lysate at 20 µg

Predicted band size: 60 kDa

Observed band size: 70 kDa,75 kDa

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• WB

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Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

All lanes:

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/2000 dilution

All lanes:

Mouse liver lysate at 10 µg

Secondary

All lanes:

goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 60 kDa

Observed band size: 80 kDa

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• WB

Lab

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (ab52629) at 1/1000 dilution

Lane 1:

Mouse liver lysate at 20 µg

Lane 2:

Rat liver lysate at 20 µg

Secondary

All lanes:

Anti-rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 60 kDa

Observed band size: 80 kDa

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• WB

CiteAb

Western blot - Anti-Scavenging Receptor SR-BI antibody [EP1556Y] (AB52629)

Western Blotting using Anti-Scavenging Receptor SR-BI antibody [EP1556Y], ab52629. Publication image from Mizusawa, H. et al., 2015, Nat Commun, 26258894. Legend direct from paper.

Gene-silencing effect with Toc-HDO targeting scavenger receptor class B member 1 (Srb1) in the liver.(a) qRT–PCR analysis showing Srb1 mRNA levels in liver assayed 3 d after injection of ASO or Toc-HDO-targeting Srb1, shuffle sequence of Toc-HDO-targeting Srb1 or PBS alone. Data are expressed as mean values±s.e.m. (n=3, **P<0.01 versus PBS). (b) Western blot analysis of Srb1 protein in livers from three mice 7 d after injection of 6 mg kg−1 Toc-HDO-targeting Srb1 mRNA. (c) qRT–PCR analyses showing Srb1 mRNA levels in liver assayed 3 d after injection of DNA/2′-MOE gapmer ASO, Toc-HDO or PBS alone. Data are expressed as mean values±s.e.m. (n=4, **P<0.01 versus PBS). (d) qRT–PCR analyses showing Srb1 mRNA levels in liver assayed 3 d after injection of mice with DNA/cEt gapmer ASO, Toc-HDO or PBS alone. Data are expressed as mean values±s.e.m. (n=4, **P<0.01 versus PBS). Data are representative of at least three independent experiments each (a,c,d). P values were calculated from the Student's two-tailed t-test (a,c,d).

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