重组Anti-Vitamin D Receptor抗体[EPR4552] - ChIP Grade

• IP

Lab

Immunoprecipitation - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Lane 1 (input) : T-47D (Human ductal breast epithelial tumor epithelial cell) whole cell lysate, 10 μg
Lane 2(+) : T-47D whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab109234 in T-47D whole cell lysate

ab109234 immunoprecipitating Vitamin D receptor in T-47D whole cell lysates. Capture antibody was used at a 1/30 dilution (2 μg in 0.35 mg lysates). For western blotting, primary antibody was used as ab109234 at 1/1000 dilution (0.62 μg/mL). VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

All lanes:

Immunoprecipitation - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234)

Predicted band size: 48 kDa

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• ChIP

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ChIP - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Chromatin was prepared from T-47D cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25 μg of chromatin, 5 μg of ab109234 (red), and 20 μL of protein A/G sepharose beads slurry (10 μL of sepharose A beads + 10 μL of sepharose G beads). 5 μg of rabbit normal IgG was added to the beads as a control sample (grey). The immunoprecipitated DNA was quantified by real time PCR (SYBR Green chemistry) with primers to c-Fos.

• WB

Lab

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

ab109234 Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade was shown to specifically react with Vitamin D Receptor in HeLa wild-type cells (ab255928). Loss of signal was observed when knockout cell line ab265430 (knockout cell lysate ab257796) was used. Wild-type and Vitamin D Receptor knockout samples were subjected to SDS-PAGE. ab109234 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234) at 1/1000 dilution

Lane 1:

Wild-type HeLa lysate at 20 µg

Lane 2:

Vitamin D Receptor knockout HeLa lysate at 20 µg

Lane 2:

Western blot - Human VDR (Vitamin D Receptor) knockout HeLa cell line (<a href='/products/cell-lines/human-vdr-vitamin-d-receptor-knockout-hela-cell-line-ab265430'>ab265430</a>)

Lane 3:

U-937 lysate at 20 µg

Lane 4:

SH-SY5Y lysate at 20 µg

Predicted band size: 48 kDa

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• WB

Lab

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Western blot : Anti-VDR antibody [EPR4552] (ab109234) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab109234 was shown to bind specifically to VDR. A band was observed at 48 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in VDR knockout cell line. To generate this image, wild-type and VDR knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

VDR knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type A549 ab288558 cell lysate at 20 µg

Lane 4:

VDR knockout A549 ab288954 cell lysate at 20 µg

Lane 5:

Wild-type HeLa ab255929 cell lysate at 20 µg

Lane 6:

VDR knockout HeLa <a href='/products/cell-lines/human-vdr-vitamin-d-receptor-knockout-hela-cell-line-ab265430'>ab265430</a> cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 48 kDa

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• WB

Unknown

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234) at 1/5000 dilution

All lanes:

HeLa cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa

Observed band size: 48 kDa

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• WB

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Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234) at 1/5000 dilution

All lanes:

Mouse kidney tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa

Observed band size: 48 kDa

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• WB

Unknown

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234) at 1/1000 dilution

All lanes:

Rat kidney tissue lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/1000 dilution

Predicted band size: 48 kDa

Observed band size: 48 kDa

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• WB

Unknown

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

All lanes:

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (ab109234) at 1/1000 dilution

Lane 1:

T47D cell lysate at 10 µg

Lane 2:

SKBR-3 cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 48 kDa

Observed band size: 48 kDa

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• WB

CiteAb

Western blot - Anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade (AB109234)

Vitamin D Receptor western blot using anti-Vitamin D Receptor antibody [EPR4552] - ChIP Grade ab109234. Publication image and figure legend from Chen, D. P., Ma, Y. P., et al., 2018, Oncotarget, PubMed 29416624.

ab109234 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab109234 please see the product overview.

1,25(OH)2 D3 modulates RMCs proliferation induced by high glucose via the DDIT4/mTOR signaling pathwayRMCs were cultured for 48 h in the presence or absence of 1,25(OH)2 D3. Cell lysates were subjected to Western blot analysis to measure the levels of the indicated proteins. The results are expressed as the relative intensity (adjusted to that of β-actin). *p < 0.05, HG vs. LG (n = 3); #p < 0.05, HG+VD vs. HG (n = 3). LG : 5.5 mM glucose; Man : 5.5 mM glucose + 24.5 mM mannitol; HG : 30 mM glucose.

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