Anti-UBP43/USP18 antibody [EPR26174-540]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-UBP43/USP18 antibody [EPR26174-540] (AB325178)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Parental A549 (human lung carcinoma epithelial cell); USP18 KO A549 (USP18 knockout human lung carcinoma epithelial cell) (ab267177); USP18 KO A549 treated with human IFNa (100 U/mL) for 14 hr cells labelling UBP43/USP18 with ab325178 at 1/100 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green).

Confocal image showing cytoplasmic and nuclear staining in Parental A549 cells (shown in green) treated with human IFNa (100 U/mL) for 14 hr. The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab7291 Anti-alpha Tubulin mouse monoclonal antibody was used to counterstain tubulin at 1/1000 dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-UBP43/USP18 antibody [EPR26174-540] (AB325178)

UBP43/USP18 was immunoprecipitated from 0.35 mg Wide-type A549 treated with 100U/ml IFN alpha (human) for 14 hours whole cell lysate with ab325178 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab325178 at 1000 dilution.

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

All lanes:

Immunoprecipitation - Anti-UBP43/USP18 antibody [EPR26174-540] (ab325178) at 1/1000 dilution

Lane 1:

Wide-type A549 treated with 100U/ml IFN alpha (human) for 14 hours whole cell lysate at 10 µg

Lane 2:

ab325178 at 1/30 IP in Wide-type A549 treated with 100U/ml IFN alpha (human) for 14 hours whole cell lysate

Lane 3:

Immunoprecipitation - Rabbit IgG, monoclonal [EPR25A] - Isotype Control (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>)

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Observed band size: 35 kDa,37 kDa

false

Exposure time: 24s

• WB

Supplier Data

Western blot - Anti-UBP43/USP18 antibody [EPR26174-540] (AB325178)

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

Performed under reducing conditions.

In Western blot, ab325178 was shown to bind specifically to UBP43/USP18. Target of interest was observed at 35 kDa and 37kDa in wide-type A549 cell lysates (lane 2) with no signal observed at this size in USP18 knockout cell line (lane 3, 4) (lane 3, knockout cell line ab267178 / knockout cell lysate ab258752).

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-UBP43/USP18 antibody [EPR26174-540] (ab325178) at 1/1000 dilution

Lane 1:

Untreated wide-type A549 (human lung carcinoma epithelial cell) whole cell lysate at 50 µg

Lane 2:

Wide-type A549 treated with 100U/ml IFN alpha (human) for 14 hours whole cell lysate at 50 µg

Lane 3:

Untreated USP18 knockout A549 whole cell lysate at 50 µg

Lane 4:

USP18 knockout A549 treated with 100U/ml IFN alpha (human) for 14 hours whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 35 kDa,37 kDa,124 kDa

false

Exposure time: 3s

• WB

Supplier Data

Western blot - Anti-UBP43/USP18 antibody [EPR26174-540] (AB325178)

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

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-UBP43/USP18 antibody [EPR26174-540] (ab325178) at 1/1000 dilution

Lane 1:

Untreated THP-1 (human monocytic leukemia monocyte) whole cell lysate at 50 µg

Lane 2:

THP-1 treated with 1 ug/ml LPS for 8 hours whole cell lysate at 50 µg

Lane 3:

Untreated HeLa (human cervical adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 4:

HeLa treated with 10ng/ml IFN alpha (human) for 16 hours whole cell lysate at 50 µg

Lane 5:

Untreated MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 50 µg

Lane 6:

MCF7 treated with 100U/ml IFN alpha (human) for 24 hours whole cell lysate at 50 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 35 kDa,37 kDa,124 kDa

false

Exposure time: 6s

• WB

Supplier Data

Western blot - Anti-UBP43/USP18 antibody [EPR26174-540] (AB325178)

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

In Western blot, Anti-Vinculin antibody [EPR8185] (ab129002) staining at 1/10000 dilution.

All lanes:

Western blot - Anti-UBP43/USP18 antibody [EPR26174-540] (ab325178) at 1/1000 dilution

All lanes:

Human tonsil tissue lysate at 50 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Observed band size: 35 kDa,37 kDa,124 kDa

false

Exposure time: 180s