I just use IL6 to stimulate the STAT3 pathway in cells. To enrich the pSTAT3 in sample, I would suggest to use nuclear extract instead of total lysate. There are kits available (e.g. Cayman 10009277) in the market. Protease and phosphatase inhibitors are included in the kit already.

Hi Blandine, 

For pancreatic islets preparation I usually use this extraction buffer (that contain thioureia and ureia to improve islet lysis):

Islets Extraction Buffer 

For 20ml : 

-EDTA 500uL 

-Urea 8,41g 

-Thiourea 3,05g 

-NaF ( 100 mM ) 84mg 

-Pyrophosphate  ( 100 mM ) 90mg 

Make up to 20 ml with water miliQ . 

Make aliquots of 1 mL and add: 

-Orthovanadate ( 10mM) 37,8mg in 200uL of miliQ water and add 10uL in 1 ml of buffer  

-PMSF 9mg in 1000uL of ethanol and add 50uL in 1 ml buffer. 

-Aprotinin 1UL the concentrated solution 

-Triton (1%) 100ul of 10% triton

I add ˜20uL in 100 to 200 islets, after this you can freeze (-80) or do protein quantification and add laemlli to the following steps to do western blotting. 

For INS1E cells you can use the same buffer or the regular RIPA buffer.

Always before protein quantification I sonicated the islets or cells to improve the protein extraction.

To stimulate STAT3 I also use just IL-6, be careful cause IL1B, TNFa and INFg increase apoptosis in beta cell.

I recommend you to use 15 wells gel and 20ug ptn per well. A important thing is your ECL buffer (I use this one: Pierce™ ECL Western Blotting Substrate 32106). I always use BSA instead of milk to all blotting steps (block, incubation antibody, etc).

ps. after use your antibody (1:1000 5%BSA) you can freeze it and use again.

Thanks a lot for your answers Bill and Nayara !

I managed to stimulate and to detect STAT3 phosphorylation with my cytokine cocktail after 2h of treatment (so I don't know if it's really a direct effect) in INS-1 cells.

We just have problems in islet, we detect phospho-STAT3 but the problem is that we have already a quite strong phosphorylation in untreated islets....So we didn't managed to induce more phosphoSTAT3 with our treatment.

I think it's because of manipulation during the different incubation and washing steps. Even if we tried to limit islet pipeting and to be very soft when we have to pipet them.

We put them for 2h in ependorfs (leaving them in the same culture medium they were already in), the supernatant is aspirated slowly (without centrifugation) and the medium with the differents treatments for 30min or 2H is added very slowed (trying not to resuspend them). Then, incubation medium is slowly aspirated before slowly adding 1mL of 4°C PBS. One centrifugation (1min30sec 1000rpm 4°C) is done, and we tried or not to do another washing step with centrifugation, but it doesn't change anything.

Do you have any other tips to not induce phosphorylation of STAT3 in untreated cells?

Thanks you in advance for your help !