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2 protocols using mouse anti cdh2

1

Western Blot Analysis of Cell Signaling

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Immunoblot analysis was performed
as previously described58 (link)−60 (link) using mouse anti-β-Actin,
mouse anti-p-ERK, rabbit anti-ERK, mouse anti-p-EGFR (Y1045), mouse
anti-p-EGFR (Y1148), mouse anti-p-EGFR (Y1173), mouse anti-CCND1,
mouse anti-CDK4, mouse anti-CCNB1, rabbit anti-c-MYC, mouse anti-BCL2,
mouse anti-BCL-XL, mouse anti-BAD, rabbit anti-CYCS, mouse anti-p-ERK
(44/42), and rabbit anti-ERK antibodies were procured from Santa Cruz
Biotechnology, CA. Mouse anti-CDH1, mouse anti-CDH2, rabbit anti-OCLN,
rabbit anti-pSTAT3, and mouse anti-STAT3 antibodies were obtained
from Abcam, Cambridge, MA. Rabbit anti-p-EGFR (Y992) and rabbit anti-p-CDK2
antibodies were obtained from Cell Signaling. Cell extracts were resolved
on SDS-PAGE and immunoblotted, with the appropriate and respective
antibodies. β-Actin was used as input control for cell lysate.
The sizes of the detected protein bands are shown in kilodaltons on
the left side.
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2

Immunoblot and Confocal Microscopy Protocol

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Immunoblot analysis was performed as previously described [23 (link)], using rabbit anti-TFF3 antibody [14 (link)]. Mouse anti-β-ACTIN, rabbit anti-p-c-SRC, mouse anti-c-SRC, and mouse anti-γ-CTNNG antibody was obtained from Santa Cruz Biotechnology, Santa Cruz, CA, USA. Mouse anti-CDH1, mouse anti-CDH2, rabbit anti-OCLN, mouse anti-VIM, mouse anti- ITGA6, rabbit anti-pSTAT3, and mouse anti-STAT3 antibody was obtained from Abcam, Cambridge, MA, USA. Cell extracts were resolved by SDS-PAGE and immunoblotted, with the respective antibodies, as previously described [23 (link)]. β-ACTIN was used as input control for cell lysate. The sizes of detected protein bands in kDa are shown on the left side.
Confocal laser scanning microscopy was performed as previously described [22 (link)]. Nuclei were visualised with VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were captured using a confocal microscope (Eclipse C1 Plus Confocal; Nikon Instrument Inc., Melville, NY, USA). Primary antibody, Mouse anti-CDH1, and mouse anti-VIM was obtained from Abcam. Secondary antibody, Alexa Fluor 488 rabbit anti-mouse immunoglobulin (Ig)G and Alexa Fluor 568 rabbit anti-mouse IgG was obtained from Invitrogen, Singapore. Rhodamine-conjugated phalloidin (Sigma-Aldrich, St Louis, MO, USA) was used to visualize f-actin filaments.
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