All lanes : Anti-DYNLL1/PIN antibody [EP1660Y] (ab51603) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : DYNLL1 knockout HeLa cell lysate
Lane 3 : Mouse testis tissue 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: 10 kDa
Observed band size: 10 kDa

Lanes 1-3: Merged signal (red and green). Green - ab51603 observed at 10 kDa. Red - loading control ab8245 observed at 36 kDa.

ab51603 Anti-DYNLL1/PIN antibody [EP1660Y] was shown to specifically react with DYNLL1/PIN in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265265 (knockout cell lysate ab257414) was used. Wild-type and DYNLL1/PIN knockout samples were subjected to SDS-PAGE. ab51603 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-DYNLL1/PIN antibody [EP1660Y] (ab51603) at 1/10000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Mouse testis lysates
Lane 3 : Rat testis lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 10 kDa
Observed band size: 10 kDa

Blocking and diluting buffer: 5% NFDM/TBST

ab51603 (purified) at 1:30 dilution (2ug) immunoprecipitating DYNLL1/PIN in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.

Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab51603 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab51603 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunocytochemistry/ Immunofluorescence analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling DYNLL1/PIN with Purified ab51603 at 1:100 dilution (6.7μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast carcinoma tissue sections labeling DYNLL1/PIN with Purified ab51603 at 1:500 dilution (1.34 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-DYNLL1/PIN antibody [EP1660Y] (ab51603) at 1/5000 dilution (unpurified)

Lane 1 : Primary mouse Mb1-Cre control Eµ-Myc B cell lymphoma (lysate of whole lymphnode)
Lane 2 : Primary mouse Mb1-Cre DYNLL1/PIN-conditional knockout Eµ-Myc B cell lymphoma (lysate of whole lymphnode)

Secondary
All lanes : HRP conjugated polyclonal goat IgG at 1/5000 dilution

Performed under reducing conditions.

Predicted band size: 10 kDa
Observed band size: 10 kDa


Exposure time: 10 minutes

Lymphnodes were dissociated in PBS 2% FBS. Cell suspensions filtered through 70 µm and 40 µm cell strainers, and 300 x g pellets were lysed in modified RIPA buffer (150 mM NaCl, 20 mM Tris pH7.4, 1 mM EDTA, 1 mM EGTA, 10 mM NaF, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 x protein inhibitor cocktail (Sigma)).

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Immunohistochemical staining of paraffin embedded human liver using unpurified ab51603 (1/100).

Flow cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling DYNLL1/PIN (red) with purified ab51603 at a 1/2300 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730). Blue (unlabeled control) - Cells without incubation with the primary and secondary antibodies.

Anti-DYNLL1/PIN antibody [EP1660Y] (ab51603) at 1/10000 dilution (unpurified) + HeLa cell lysate at 10 µg

Secondary
Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 10 kDa
Observed band size: 10 kDa

Unpurified ab51603 staining DLC8 in mouse kidney cells cells by ICC/IF (immunocytochemistry/immunofluorescence. Cells were fixed with methanol, permeabilized with 0.1% Triton and blocked with 1% milk for 1 hour at room temperature. The sample was incubated with primary antibody (1/400; 1% milk in PBS) for 16 hours at 4°C. An Alexa Fluor^®488-conjugated Goat polyclonal to rabbit IgG (1/1000) was used as secondary antibody.

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Unpurified ab51603 used in IP.SKAP and Astrin form a complex. (A, left) Silver-stained gels showing a one-step IP of GFPLAP-Astrin, GFPLAP-SKAP, or GFPLAP-LC8. (A, right) Data from the mass spectrometric analysis of the purifications indicating the percent sequence coverage from each IP. (B) Silver-stained gel showing the purification of FLAG-SKAP from chicken DT40 cells relative to controls. The indicated proteins were identified by excising them from a gel and analyzing them by mass spectrometry.