Figure 4. RHOA G17V interacts with p300... | American Society of Hematology

Figure 4.

$RHOA G17V interacts with p300 and both colocalize. (A) RHOA G17V interacts with p300 in modified Jurkat cells. Representative western blot analysis of lysates (left) and anti-HA IP of ectopically expressed HA-tagged RHOA (right) for the indicated cell lines. Cells were costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 5 minutes. Whole-cell lysates (input, left) and (IP, right) were analyzed by immunoblotting (IB) using anti-HA, anti–p-VAV1, anti-VAV1, anti-VAVA3, and anti-p300 antibodies. Positions of molecular weight markers are indicated in kilodalton. The experiment was performed 3 times. (B) RHOA G17V interacts with p300 at every time point of costimulation. Representative western blot analysis of lysates (left) and anti-HA IPs of ectopically expressed HA-tagged RHOA (right) from the indicated cell lines. Cells were costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL during the indicated periods of time. Whole-cell lysates (input, left) and IPs (right) were analyzed by IB using anti-HA, anti-GAPDH, and anti-p300 antibodies. The experiment was performed 3 times. (C) RHOA G17V interacts with p300 in human primary T cells. Representative western blot analysis of lysates (left) and anti-HA IPs of ectopically expressed HA-tagged RHOA wt or G17V (right) from human primary T cells. Whole-cell lysates (input, left) and IPs (right) were analyzed by IB using anti-HA, anti-APDH, and anti-p300 antibodies. Positions of molecular weight markers are indicated in kilodalton. The experiment was performed twice. (D) Representative western blot analysis of cytoplasmic (left) and nuclear (right) cellular fractions of cell lines expressing ectopically either the wt or G17V form of RHOA. Cells were costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL during the indicated time periods. Whole-cell lysates were analyzed by IB using the indicated antibodies. IGF1R and Lamin B1 immunoblots show the separation between cytoplasmic and nuclear fractions. The experiment was performed twice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.$

RHOA G17V interacts with p300 and both colocalize. (A) RHOA G17V interacts with p300 in modified Jurkat cells. Representative western blot analysis of lysates (left) and anti-HA IP of ectopically expressed HA-tagged RHOA (right) for the indicated cell lines. Cells were costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL for 5 minutes. Whole-cell lysates (input, left) and (IP, right) were analyzed by immunoblotting (IB) using anti-HA, anti–p-VAV1, anti-VAV1, anti-VAVA3, and anti-p300 antibodies. Positions of molecular weight markers are indicated in kilodalton. The experiment was performed 3 times. (B) RHOA G17V interacts with p300 at every time point of costimulation. Representative western blot analysis of lysates (left) and anti-HA IPs of ectopically expressed HA-tagged RHOA (right) from the indicated cell lines. Cells were costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL during the indicated periods of time. Whole-cell lysates (input, left) and IPs (right) were analyzed by IB using anti-HA, anti-GAPDH, and anti-p300 antibodies. The experiment was performed 3 times. (C) RHOA G17V interacts with p300 in human primary T cells. Representative western blot analysis of lysates (left) and anti-HA IPs of ectopically expressed HA-tagged RHOA wt or G17V (right) from human primary T cells. Whole-cell lysates (input, left) and IPs (right) were analyzed by IB using anti-HA, anti-APDH, and anti-p300 antibodies. Positions of molecular weight markers are indicated in kilodalton. The experiment was performed twice. (D) Representative western blot analysis of cytoplasmic (left) and nuclear (right) cellular fractions of cell lines expressing ectopically either the wt or G17V form of RHOA. Cells were costimulated with anti-CD3, anti-CD28, and crosslinker at 2 μg/mL during the indicated time periods. Whole-cell lysates were analyzed by IB using the indicated antibodies. IGF1R and Lamin B1 immunoblots show the separation between cytoplasmic and nuclear fractions. The experiment was performed twice. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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