All lanes : Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SESN2 knockout HeLa cell lysate
Lane 3 : LoVo cell lysate
Lane 4 : HEK-293T 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: 54 kDa
Observed band size: 54 kDa

Lanes 1-4: Merged signal (red and green). Green - ab178518 observed at 54 kDa. Red - loading control ab8245 observed at 36 kDa.

 ab178518 Anti-SESN2/Sestrin-2 antibody [EPR18907] was shown to specifically react with SESN2/Sestrin-2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265669 (knockout lysate ab257665) was used. Wild-type and SESN2/Sestrin-2 knockout samples were subjected to SDS-PAGE. ab178518 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2.5 hours 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HCT 116 (Human colorectal carcinoma cell line) cells labeling SESN2/Sestrin-2 with ab178518 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 HCT116 cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594)  at 1/250 dilution (red).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution.

All lanes : Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : SESN2 knockout HeLa cell lysate
Lane 3 : HeLa 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: 54 kDa
Observed band size: 54 kDa

Lanes 1-3: Merged signal (red and green). Green - ab178518 observed at 54 kDa. Red - loading control ab8245 observed at 36 kDa. 

 ab178518 Anti-SESN2/Sestrin-2 antibody [EPR18907] was shown to specifically react with SESN2/Sestrin-2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265669 (knockout cell lysate ab257665) was used.  Wild-type and SESN2/Sestrin-2 knockout samples were subjected to SDS-PAGE.  ab178518 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 : Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1 : Untreated HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate treated with 10 mM H2O2 for 1 hour
Lane 3 : LoVo (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 4 : 293T (Human epithelial cell line from embryonic kidney) whole cell lysate
Lane 5 : NIH/3T3 (Mouse embryonic fibroblast cell line) 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: 54 kDa
Observed band size: 54 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Sestrin expression is induced by H₂O₂ treatment, which is consistent with what has been described in the literature (PMID: 25337554).

Exposure time:3 minutes

All lanes : Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1 : Human colon lysate
Lane 2 : Human fetal liver lysate
Lane 3 : Human testis lysate
Lane 4 : Human fetal kidney lysate

Lysates/proteins at 20 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: 54 kDa
Observed band size: 54 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time:3 minutes

All lanes : Anti-SESN2/Sestrin-2 antibody [EPR18907] (ab178518) at 1/1000 dilution

Lane 1 : HCT 116 (Human colorectal carcinoma cell line) whole cell lysate
Lane 2 : Rat1 (Rat fibroblast cell line) whole cell lysate
Lane 3 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 4 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 5 : Mouse spleen lysate
Lane 6 : PC-12 (Rat adrenal gland pheochromocytoma cell line) 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: 54 kDa
Observed band size: 54 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1, 2, 3, 4 and 5: 3 minutes; Lane 6: 30 seconds.

SESN2/Sestrin-2 was immunoprecipitated from 0.35 mg of HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab178518 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab178518 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: ab178518 IP in HeLa whole cell lysate.

Lane 3: Rabbit IgG,monoclonal [EPR25A] - Isotype Control (ab172730) instead of ab178518 in HeLa whole cell lysate.

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

Exposure time: 3 minutes.

SESN2/Sestrin-2 expression is low in HeLa cells and can be enriched through immunoprecipitation.

Flow cytometric analysis of 4% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/60 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.

Flow cytometric analysis of 4% paraformaldehyde-fixed HCT 116 (Human colorectal carcinoma cell line) cells labeling SESN2/Sestrin-2 with ab178518 at 1/60 dilution (red) compared with a rabbit monoclonal IgG 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.