Anti ps727 stat3

Flag-CREPT, Myc-CREPT, Myc-CREPT/RPR, Myc-CREPT/CCT, Flag-STAT3, APRE (acute phase response element)-luciferase and pCDH-HA-CREPT were constructed in this laboratory. HA-p300 plasmid was a gift from Dr. Y. Eugene Chin (Institutes of Biology and Medical Sciences, Soochow University). Anti-STAT3 (c-20), anti-Myc (9E10) and anti-HA (F-7) were purchased from Santa Cruz Biotechnology (Santa Cruz). Anti-p300 (D2X6N), anti-c-MYC, anti-cyclinD1 (SP4), anti-pY705 STAT3 (D3A7), anti-pS727 STAT3, anti-acK685 STAT3, anti-Bcl-XL (54H6), anti-H3K18ac (D8Z5H) and anti-H3K27ac (D5E4) antibodies were purchased from Cell Signaling Technology. Anti-actin (AC-15) and anti-Flag (M2) antibodies were purchased from Sigma. Anti-CREPT antibody was prepared by this laboratory.^{50 (link)} The cytokine leukaemia inhibitory factor (LIF) was purchased from Millipore (cat. #LIF1010). Short interfering RNAs (siRNAs) against CREPT or p300 were synthesised from GenePharma (SuZhou GenePharma Co. Ltd) with the oligo sequence information as shown in Table S1. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9)-mediated CREPT deletion plasmids were generated based on PX458M vector with guider RNAs and the sequence information was shown in Table S1.

After stimulation with rmIL-33 and/or rmSCF (both 50 ng/mL) (Peprotec), BMMCs were lysed in lysis buffer (20 mM HEPES, pH 7.5; 10 mM EGTA, 40 mM β−glycerophosphate, 2.5 mM MgCl₂, 2 mM orthovanadate, 1 mM dithiothreitol, 20 µg/mL aprotinin, and 20 µg/mL leupeptin supplemented with 1% Triton). The protein concentration was determined (BCA−kit; Pierce), and the samples were boiled (with 6 × Laemmli buffer). The lysates were separated with 10% sodium dodecyl sulfate (SDS) −Laemmli gels and afterwards transferred onto nitrocellulose membranes (biostep) by Western blotting. The membranes were blocked (with dry milk) and incubated with anti-pT180/pY182−p38, anti-p38, anti-pS177/181−IKK2, anti-p184/187−pTAK1, anti-pT202/pY204−ERK1/2, anti-pS32−IκBα, anti-IκBα, anti-pS536−p65, anti-p65, HSP90, anti-pST183/Y185−JNK1/2, anti-JNK1/2, anti-pS727−STAT3, anti-STAT3, anti-pS235/236−S6, anti-S6, and anti-tubulin (all from Cell Signaling except anti-IKK1/2, anti-TAK1, and anti-ERK1/2 (Santa Cruz, CA, USA)). The membranes were washed with TBS/0.1% Tween and incubated with HRP-conjugated secondary anti-rabbit Ig, anti-mouse Ig, or anti-goat Ig (SeraCare). We used ECL reagent (Pierce) for detection.

Sodium bicarbonate (NaHCO₃), lactic acid, sodium lactate and sodium oxamate were obtained from Sigma Aldrich (St. Louis, MO, USA). The antibodies used in this study were as follows: anti-PD-L1 (Abcam, Cambridge, UK); anti-LDH-A (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-pY⁷⁰¹-STAT1, anti-STAT1, anti-pY⁷⁰⁵-STAT3, anti-pS⁷²⁷-STAT3, anti-STAT3 and anti-β-actin (Cell Signaling Technology, Danvers, MA, USA); and anti-HIF-1α (Abbkine, Wuhan, China).