FAK (phospho Tyr397) antibody

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-Store as concentrated solution. Centrifuge briefly prior to opening vial.For short-termstorage(1-2 weeks), storeat 4ºC.For long-term storage, aliquot and storeat -20ºC or below. Avoid multiple freeze-thaw cycles.	-Rabbit (1:1000)

Upstream tips
-Store as concentrated solution. Centrifuge briefly prior to opening vial.For short-termstorage(1-2 weeks), storeat 4ºC.For long-term storage, aliquot and storeat -20ºC or below. Avoid multiple freeze-thaw cycles.
Protocol tips
-Rabbit (1:1000)

Publication protocol

Cells were lysed and protein was extracted using M-PER (Pierce, Rockford, IL) plus protease inhibitor cocktail (Pierce), with protein concentrations being determined using the BCA assay (Pierce). Aliquots of protein lysates were separated on SDS-10% polyacrylamide gels and transferred on polyvinylidenedifluoride membrane, which was blocked with 5% milk (Bio-Rad, Richmond, CA) in TBST. The membrane was then hybridized with primary antibodies, followed by corresponding secondary antibodies and then detected using a chemiluminescence assay (Millipore, Billerica, MA). Membranes were exposed to an X-ray film to visualize the bands (Amersham Pharmacia Biotech, Piscataway, NJ). Antibodies against pSTAT3 (S727; catalogue No. 9136; 1:1,000), pSTAT3 (Y705; catalogue No. 9131; 1:1,000), STAT3 (catalogue No. 9132; 1:2,000), PP2A C subunit (catalogue No. 4957; 1:2,000), pAkt (S473; catalogue No. 9271; 1:1,000), β-tubulin (catalogue No. 2146; 1:2,000), Flag (catalogue No. 2368; 1:2,000), pAkt (T308; catalogue No. 9275; 1:1,000), Akt (catalogue No. 9272; 1:2,000), ERK (catalogue No. 9102; 1:2,000), pERK (catalogue No. 9101; 1:1,000), pP38 (T180 and Y182; catalogue No. 9211; 1:1,000), P38 (catalogue No. 9212; 1:2,000), pJNK (catalogue No. 9251; 1:1,000) and pmTOR (S2448; catalogue No. 2976; 1:1,000) were purchased from Cell Signaling Technologies. Antibodies against pPP2A (Y307; catalogue No. sc-12615; 1:2,000), Oct4 (catalogue No. sc-9081; 1:500), Nanog (catalogue No. sc-30328; 1:500), Histone H1 (catalogue No. sc-8030; 1:1,000) and Sox2 (catalogue No. sc-20088; 1:500) were purchased from Santa Cruz Biotechnology Inc. Antibodies against pFAK (Y397) (catalogue No. GTX24803; 1:1,000) were purchased from GeneTex (San Antonio, TX). Antibody against Col17a1 (catalogue No. ab28440; 1:500) was purchased from Abcam. Antibody against Laminin γ2 (catalogue No. MAB19562; 1:1,000) was purchased from Chemicon (Temecula, CA). Antibody against cleaved-caspase3 (catalogue No. 1476-1; 1:1,000) was purchased from Epitomics (Burlingame, CA). All the experiments of western blot analysis presented with representative images were repeated at least twice.

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Manufacturer protocol

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