All lanes : Anti-DR5 antibody [EPR19310] (ab199357) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : DR5 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 48 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab199357 observed at 47 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab199357 was shown to react with DR5 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264922 (knockout cell lysate ab257748) was used. Wild-type HeLa and DR5 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab199357 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 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.

Flow cytometric analysis of 4% paraformaldehyde-fixed HCT 116 (Human colorectal carcinoma cell line) cells labeling DR5 with ab199357 at 1/70 dilution (red) compared with a Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody; blue). Goat anti Rabbit IgG (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HT1080 (Human fibrosarcoma cell line) cells labeling DR5 with ab199357 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HT1080  cells. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab199357 at 1/100 dilution followed by ab150120 at 1/1000 dilution.
-ve control 2: ab7291 at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

All lanes : Anti-DR5 antibody [EPR19310] (ab199357) at 1/1000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : TNFRSF10B knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 48 kDa
Observed band size: 47 kDa why is the actual band size different from the predicted?

Lanes 1 - 2: Merged signal (red and green). Green - ab199357 observed at 47 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab199357 was shown to recognize DR5 in wild-type HAP1 cells as signal was lost at the expected MW in TNFRSF10B knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and TNFRSF10B knockout samples were subjected to SDS-PAGE. Ab199357 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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 : Anti-DR5 antibody [EPR19310] (ab199357) at 1/1000 dilution

Lane 1 : Untreated HCT 116 (Human colorectal carcinoma cell line) whole cell lysate
Lane 2 : HCT 116 (Human colorectal carcinoma cell line) treated with 0.5µM/ml doxorubicin for 24 hours whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48 kDa
Observed band size: 40,48 kDa why is the actual band size different from the predicted?


Exposure time: 8 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Doxorubicin treatment elevated the expression of DR5 (PMID: 12496481; PMID: 11468181; PMID: 11090076). The expression profile is consistent with what has been described in the literature (PMID:20515924; PMID:16297203).

All lanes : Anti-DR5 antibody [EPR19310] (ab199357) at 1/1000 dilution

Lane 1 : Human melanoma lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 4 : HT1080 (Human fibrosarcoma cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 48 kDa
Observed band size: 40,48 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1-2: 3 minutes; Lane 3: 10 seconds; Lane 4: 8 seconds.

The expression profile is consistent with what has been described in the literature (PMID:20515924; PMID:16297203).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling DR5 with ab199357 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous and cytoplasmic staining on HCT 116 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab199357 at 1/100 dilution followed by ab150120  at 1/1000 dilution.
-ve control 2: ab7291  at 1/1000 dilution followed by ab150077  at 1/1000 dilution.

DR5 was immunoprecipitated from 0.35mg of HT1080 (Human fibrosarcoma cell line) treated with 5μM MG132 for 4 hour whole cell lysate with ab199357 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab199357 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HT1080 treated with 5μM MG132 for 4 hour whole cell lysate, 10µg (Input).

Lane 2: ab199357 IP in HT1080 treated with 5μM MG132 for 4 hour whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab199357 in HT1080 treated with 5μM MG132 for 4 hour whole cell lysate.

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

Exposure time: 10 seconds.

DR5 was immunoprecipitated from 0.35mg of HCT 116 (Human colorectal carcinoma cell line) whole cell lysate with ab199357 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab199357 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HCT 116 whole cell lysate, 10µg (Input).

Lane 2: ab199357 IP in HCT 116 whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730)  instead of ab199357 in HCT 116 whole cell lysate.

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

Exposure time: 10 seconds.