P stat3 ser727

Cells were lysed with RIPA buffer containing a proteinase inhibitor cocktail (Fisher Scientific, Pittsburg, PA), sheared 10 times by passage through a 28-gauge needle, and centrifuged at 16,000 g for 30 min; the supernatants were normalized for protein concentration as determined by the Bradford method. Lysates were boiled for 5 min and resolved on 4–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels (Invitrogen, Carlsbad, CA). The blots were probed with the following primary antibodies from Cell Signaling Technology (Danvers, MA): p-STAT3-Tyr705, p-STAT3-Ser727, HKII, P70, and P70S6K. The following antibodies were from Santa Cruz Biotechnology (Dallas, TX): anti-actin, anti-Prx1 and anti-Prx3.

Protein concentrations were measured using the Micro BCA™ Protein Assay Kit (23235; Thermo Fisher) and proteins were resolved by 10% or 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Amersham Pharmacia, Uppsala, Sweden). The levels of HIF-1α (ab179483; Abcam), signal transducer and activator of transcription 3 (STAT3; #9139; Cell Signaling Technology, Danvers, MA, USA), p-STAT3^Tyr705 (#9145; Cell Signaling Technology), p-STAT3^Ser727 (#9136; Cell Signaling Technology), lysosomal-associated membrane protein 1 (LAMP1; sc-20011; Santa Cruz Biotechnology, Santa Cruz, TX, USA), p62 (ab56416; Abcam), microtubule-associated protein 1 light chain 3 (LC3; ab48394; Abcam), p-MLKL (ab184718; Abcam), MLKL (Abcam), and GAPDH (ab181602; Abcam) were detected by Western blotting. Signals were detected using anti-mouse or anti-rabbit HRP-conjugated secondary antibodies. The western blots were quantification using Fiji/imageJ program. Target protein was normalized with GAPDH and, phospho-proteins normalized with total protein and GAPDH.

Western blot analysis was performed in accordance to standard protocols. Total proteins were prepared from mice hearts. Total protein extracts were isolated from mice hearts with 250 mM sucrose buffer. Primary antibodies against AKT (1 : 1000), p-AKT (Ser-473, 1 : 1000), mTOR (1 : 1000), p-mTOR (Ser2448, 1 : 1000), PTEN (1 : 1000), p62 (1 : 1000), LC3-II/I (1 : 1000), STAT3, p-STAT3 (Ser-727, 1 : 1000), and β-actin (1 : 2000) were purchased from Cell Signalling Technology (Beverly, MA). Band images were scanned, and densitometric analysis was performed using NIH Image software (Bethesda, MD, USA).

B cells were fixed in 3% glutaraldehyde and postfixed in 1 × PBS/1%
osmium tetroxide, dehydrated in ethanol followed by propylene oxide (VWR, Radnor, PA, USA), embedded
in Epon and polymerized overnight at 60 °C. Ultrathin sections were cut with a
LEICA/Reichert Ultracut S ultramicrotome, stained with uranyl acetate and lead citrate.
Immunolabeling was performed on B cells fixed in 0.2% glutaraldehyde/4%
paraformaldehyde for 1 h at room temperature as described,^{40 (link)} using rabbit total STAT3 or pSTAT3Ser₇₂₇ antibodies (Cell Signaling
Technology). Ultrathin (Peabody, MA, USA) sections were examined with a JEOL1011 transmission
electron microscope. Digital images of ≥10 cells/sample were obtained using a GATAN-CCD
camera and Digital Micrograph software (Warrendale, MA, USA).