All lanes : Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/1000 dilution

Lane 1 : Wild-type U-2 OS cell lysate
Lane 2 : FHL2 knockout U-2 OS cell lysate
Lane 3 : HeLa cell lysate

Lysates/proteins at 40 µg per lane.

Performed under reducing conditions.

Predicted band size: 32 kDa
Observed band size: 32 kDa

Lanes 1 - 3: Merged signal (red and green). Green - ab202586 observed at 32 kDa. Red - loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab202586 was shown to react with FHL2 in wild-type U-2 OS cells in western blot with loss of signal observed in FHL2 knockout sample. Wild-type and FHL2 knockout U-2 OS cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab202586 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

ab202586 staining FHL2 in wild-type U2OS cells (top panel) and FHL2 knockout U2OS cells (bottom panel). The cells were fixed with 4% PFA (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab202586 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

All lanes : Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/1000 dilution

Lane 1 : Wild-type HeLa lysate
Lane 2 : FHL2 knockout HeLa lysate
Lane 3 : K562 lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 32 kDa

Lanes 1-3: Merged signal (red and green). Green - ab202586 observed at 32 kDa. Red - loading control ab7291 observed at 50 kDa.

 ab202586 Recombinant Anti-FHL2 antibody [EPR17860-23] was shown to specifically react with FHL2 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265475 (knockout cell lysate ab257441) was used. Wild-type and FHL2 knockout samples were subjected to SDS-PAGE. ab202586 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.

 

ab202586 staining FHL2 in wild-type U2OS cells (top panel) and FHL2 knockout U2OS cells (bottom panel). The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab202586 at 1/100 dilution and ab7291 (Tubulin) at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to mouse IgG (Alexa Fluor® 594) (ab150120) at 2 μg/ml (shown in pseudo color red).  Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A-673 (Human muscle Ewing's Sarcoma cell line) cells labeling FHL2 with ab202586 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weakly nuclear staining on A-673 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 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab202586 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 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 antibody at 1/500 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embyro fibroblast cells) cells labeling FHL2 with ab202586 at 1/100 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/500 dilution (green). Confocal image showing cytoplasmic and weakly nuclear staining on A-673 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 1/500 dilution (red).

The negative controls are as follows:
-ve control 1: ab202586 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/500 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 antibody at 1/500 dilution.

All lanes : Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/2000 dilution

Lane 1 : SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate
Lane 2 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 3 : HT1080 (Human fibrosarcoma cells) whole cell lysate
Lane 4 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa


Exposure time: 3 minutes

5% NFDM/TBST: Blocking and diluting buffer.

Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/10000 dilution + Human fetal heart lysate at 10 µg

Secondary
Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-FHL2 antibody [EPR17860-23] (ab202586) at 1/10000 dilution

Lane 1 : Mouse heart lysate
Lane 2 : Mouse kidney lysate
Lane 3 : Rat heart lysate
Lane 4 : Rat kidney lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

Predicted band size: 32 kDa
Observed band size: 32 kDa


Exposure time: 30 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

FHL2 was immunoprecipitated from 1mg of SW480 (Human colorectal adenocarcinoma cell line) whole cell lysate with ab202586 at 1/20 dilution. Western blot was performed from the immunoprecipitate using ab202586 at 1/1000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG, was used as secondary antibody at 1/1500 dilution.

Lane 1: SW480 whole cell lysate 10 µg (Input). Lane 2: ab202586 IP in SW480 whole cell lysate. Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab202586 in SW480 whole cell lysate.

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

Exposure time: 5 seconds.