All lanes : Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : CNOT7 knockout HeLa cell lysate
Lane 3 : MOLT-4 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa

Lanes 1-3: Merged signal (red and green). Green - ab195587 observed at 33 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab195587 Anti-CNOT7 antibody [EPR18722] was shown to specifically react with CNOT7 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265811 (knockout cell lysate ab258370) was used. Wild-type and CNOT7 knockout samples were subjected to SDS-PAGE. ab195587 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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 : Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1 : Mouse ovary lysate
Lane 2 : Mouse embryo lysate
Lane 3 : F9 (Mouse embryonic testicular cancer cell line) whole cell lysate
Lane 4 : SP2/0 (Mouse spleen cell line) whole cell lysate
Lane 5 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma 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: 33 kDa
Observed band size: 33 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 2 seconds; Lane 2-6: 5 seconds.

All lanes : Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1 : Mouse brain lysate
Lane 2 : Mouse heart lysate
Lane 3 : Rat kidney lysate
Lane 4 : Rat spleen lysate
Lane 5 : Rat heart 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: 33 kDa
Observed band size: 33 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1/2: 15 seconds; Lane 3/4: 5 seconds;  Lane 5: 30 seconds.

 

All lanes : Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney lysate
Lane 4 : Human fetal spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 33 kDa
Observed band size: 33 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

 

All lanes : Anti-CNOT7 antibody [EPR18722] (ab195587) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HepG2 (Human liver hepatocellular carcinoma cell line) whole cell lysate
Lane 3 : NTERA-2/D1 (Human malignant pluripotent embryonic carcinoma cell line) whole cell lysate
Lane 4 : NCCIT (Human pluripotent embryonic carcinoma 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: 33 kDa
Observed band size: 33 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time: Lane 1: 1 seconds; Lane 2-4: 2 seconds.

 

CNOT7 was immunoprecipitated from 1mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab195587 at 1/40 dilution. Western blot was performed from the immunoprecipitate using ab195587 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: HeLa whole cell lysate 10µg (Input).

Lane 2: ab195587 IP in HeLa whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab195587 in HeLa whole cell lysate.

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

Exposure time: 1 second.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling CNOT7 with ab195587 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab195587at 1/1000 dilution, followed by Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized F9 (Mouse embryonic testicular cancer cell line) cells labeling CNOT7 with ab195587 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows:
-ve control 1: ab195587at 1/1000 dilution, followed by Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/XXXX dilution.
-ve control 2:  Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

Flow cytometry analysis of 4% paraformaldehyde fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cell line labelling CNOT7 with ab195587 at 1/1000 dilution (red), isotype control with ab172730 (black) and unlabelled control cells without incubation with primary antibody and secondary antibody (blue).

Flow cytometry analysis of 4% paraformaldehyde fixed F9 (Mouse embryonic testicular cancer cell line) cells labeling CNOT7 with ab195587 at 1/1000 dilution (red), isotype control with ab172730 (black) and unlabeled control cells without incubation with primary antibody and secondary antibody (blue).