711190

antibody from Invitrogen Antibodies

Targeting: CLDN3 C7orf1, CPE-R2, CPETR2, HRVP1, RVP1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711190

Provider

Invitrogen Antibodies

Product name

Phospho-Claudin 3 (Thr192) Recombinant Polyclonal Antibody (4HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

4HCLC

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on Membrane enriched cell extracts (30 µg lysate) of SK-OV-3 (Lane 1), SK-OV-3 treated with Forskolin and PMA (Serum starved for 16 hrs and 30 uM Forskolin and 50 ng/mL PMA for 20 min) (Lane 2), HT-29 (Lane 3) and HT-29 treated with Forskolin and PMA (Serum starved for 16 hrs and 30 uM Forskolin and 50 ng/mL PMA for 20 min) (Lane 4). The blots were probed with Anti-Claudin3 (pT192) Recombinant Rabbit Polyclonal Antibody (Product # 711190, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 35 kDa band corresponding to Claudin3 (pT192) was observed across the cell lines tested and * non specific band at 70 kDa was observed. Known quantity of protein samples were electrophoresed using Novex®NuPAGE®4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, Caco-2 cells were fixed and permeabilized for detection of endogenous Claudin3 pT192 using Anti- Claudin3 pT192 Recombinant Rabbit Polyclonal Antibody (Product # 711190, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of Claudin3 pT192 (green) in the cytoplasm can be clearly observed in cells treated with Forskolin (30 uM, 20 min) as compared to untreated cells. Antibody specificity was demonstrated by competition with the Claudin3 pT192 peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.