Human TMPRSS2 knockout LNCaP cell line

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Human TMPRSS2 knockout LNCaP cell line (AB273745)

ab280567 staining TMPRSS2 in wild-type LnCAP cells (top panel) and TMPRSS2 knockout LnCAP cells (ab273745) (bottom panel). The cells were fixed with 4% paraformaldehyde (10 min) then 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 ab280567 at 1μg/ml concentration and ab7291 (Mouse monoclonal to alpha 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 red). Nuclear DNA was labelled in blue with DAPI.
Image was taken with a confocal microscope (Leica-Microsystems TCS SP8).

• WB

Lab

Western blot - Human TMPRSS2 knockout LNCaP cell line (AB273745)

False colour image of Western blot : Anti-TMPRSS2 antibody [EPR3862] staining at 1/2000 dilution shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution shown in red.

In Western blot ab109131 was shown to bind specifically to TMPRSS2. A band was observed at 55 25 kDa in wild-type LNCaP and at 25 kDa in Caco-2 cell lysates with no signal observed at this size in TMPRSS2 knockout LNCaP cell line ab273745 (knockout LNCaP cell lysate ab275499) and TMPRSS2 knockout Caco-2 cell line ab273737 (knockout Caco-2 cell lysate ab277340).

To generate this image wild-type and TMPRSS2 knockout cell lysates were analysed. First samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °. Blots were washed four times in TBS-T incubated with secondary antibodies for 1 h at room temperature washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-TMPRSS2 antibody [EPR3862] (<a href='/products/primary-antibodies/tmprss2-antibody-epr3862-ab109131'>ab109131</a>) at 1/2000 dilution

Lane 1:

Wild-type LNCaP cell lysate at 20 µg

Lane 2:

TMPRSS2 knockout LNCaP cell lysate at 20 µg

Lane 2:

Western blot - Human TMPRSS2 knockout LNCaP cell line (ab273745)

Lane 3:

Wild-type Caco-2 cell lysate at 20 µg

Lane 4:

TMPRSS2 knockout Caco-2 cell lysate at 20 µg

Lane 5:

PC-3 cell lysate at 20 µg

Lane 6:

DU 145 cell lysate at 20 µg

Lane 7:

Human Prostate cell lysate at 20 µg

Lane 8:

Human Colon cell lysate at 20 µg

Lane 9:

Human Lung cell lysate at 20 µg

Predicted band size: 54 kDa

Observed band size: 25 kDa,55 kDa

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• WB

Supplier Data

Western blot - Human TMPRSS2 knockout LNCaP cell line (AB273745)

The samples were run on a Bis-Tris gel under reducing conditions. Western blot : Anti-TMPRSS2 antibody (ab309546) staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab309546 was shown to bind specifically to TMPRSS2. Target of interest was observed at 55 kDa and 25 KDa in wild-type LNCaP cell lysates (lane 1) with no signal observed at this size in TMPRSS2 knockout cell line (lane 2, knockout cell line ab273745). To generate this image, samples were first run on an SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in a fluorescent western blot (TBS-based) blocking solution before incubation with primary antibodies overnight at 4 °C. Blots were washed in TBS-T, incubated with secondary antibodies Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution for 1 h at room temperature, washed again then imaged. The molecular weight observed is consistent with what has been described in the literature (PMID : 35104687). Exposure time :

All lanes:

Western blot - Anti-TMPRSS2 antibody [P5H9-A3] (<a href='/products/primary-antibodies/tmprss2-antibody-p5h9-a3-ab309546'>ab309546</a>) at 1/1000 dilution

Lane 1:

Wild-type LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

TMPRSS2 knockout LNCaP whole cell lysate at 20 µg

Lane 2:

Western blot - Human TMPRSS2 knockout LNCaP cell line (ab273745)

Secondary

Lanes 1 - 2:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/200000 dilution

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/200000 dilution

Observed band size: 25 kDa,55 kDa

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• sELISA

Lab

Sandwich ELISA - Human TMPRSS2 knockout LNCaP cell line (AB273745)

Human TMPRSS2 concentration was interpolated from the TMPRSS2 standard curve. Supernatants from cell culture samples were serially diluted and assessed by the Human TMPRSS2 ELISA Kit (ab283552). Wild-type and TMPRSS2 knockout LNCaP (ab273745) cells were assessed in duplicate (n=2). Data are represented as the mean and error bars represent standard deviation.

• Sanger seq

Lab

Sanger Sequencing - Human TMPRSS2 knockout LNCaP cell line (AB273745)

79 bp deletion in exon 3