Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527]

• WB

Lab

Western blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (AB325926)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

Performed under reducing conditions.

In Western blot, aab325926 was shown to bind specifically to Cyclin D1 (phospho T286). Target of interest was observed at 33 kDa in A549 treated with 10µM MG-132 for 4 hours lysates (lane 2) with no signal observed at this size in CCND1 (Cyclin D1) knockout cell line (lanes 3-4) (lanes 3-4, knockout cell line ab286759).

The identity of the bands higher than 75kda are unknown.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (ab325926) at 1/1000 dilution

Lane 1:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate 20 µg (untreated membrane)

Lane 2:

A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (untreated membrane)

Lane 3:

Untreated CCND1 (Cyclin D1) knockout A549 whole cell lysate 20 µg (untreated membrane)

Lane 4:

CCND1 (Cyclin D1) knockout A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (untreated membrane)

Lane 5:

Untreated A549 (human lung carcinoma epithelial cell) whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 6:

A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 7:

Untreated CCND1 (Cyclin D1) knockout A549 whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Lane 8:

CCND1 (Cyclin D1) knockout A549 treated with 10µM MG-132 for 4 hours whole cell lysate 20 µg (alkaline phosphatase treated membrane)

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 33 kDa,36 kDa

false

Exposure time: 48s

• WB

Lab

Western blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (AB325926)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) (1 : 200000) (36KDa).

All lanes:

Western blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (ab325926) at 1/1000 dilution

Lane 1:

Untreated HT-1080 (human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HT-1080 treated with 10µM MG-132 for 4 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Observed band size: 33 kDa,36 kDa

false

Exposure time: 92s

• Dot

Lab

Dot Blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (AB325926)

Dot blot analysis of Cyclin D1 using ab325926 at 1 : 10000 (0.051 µg/ml) followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1 : 100,000 dilution.

Lane 1 : Human Cyclin D1 (phospho T286) peptide

Lane 2 : Human Cyclin D1 (non-phospho) peptide

Lane 3 : Human Cyclin D2 (phospho T280) peptide

Lane 4 : Human Cyclin D3 (phospho T283) peptide

Exposure time : 180 seconds.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

The antibody recognises CCND1 (Cyclin D1) when phosphorylated at Threonine 286, and shows weak reactivity with CCND2 (Cyclin D2) when phosphorylated at Threonine 280 by dot blot.

All lanes:

Dot Blot - Anti-Cyclin D1 (phospho T286) antibody [EPR30260-527] (ab325926) at 1/10000 dilution

Lane 1:

Human Cyclin D1 (phospho T286) peptide

Lane 2:

Human Cyclin D1 (non-phospho) peptide

Lane 3:

Human Cyclin D2 (phospho T280) peptide

Lane 4:

Human Cyclin D3 (phospho T283) peptide

Secondary

All lanes:

Dot Blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

false

Exposure time: 180s