Stat5a

Antibodies to phosphorylated Stat5a/Stat5B (Millipore, USA), Stat5a (Abcam, Hong Kong) were used for Western blotting. The isolated lymphocytes from lung tissues were lysed on ice in a buffer consisting of 50 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40, and complete proteinase inhibitor cocktail (Boster Biological Engineering, China). After being centrifuged, the cell lysates (30 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane (Boster). The membrane was blocked with 5% fat-free dry milk in TBST and incubated with antibodies against phosphorylated Stat5a/STAT5B (Millipore, USA), or Stat5a. The bound antibodies were detected using horseradish peroxidase- (HRP-) conjugated second antibodies and visualized using enhanced chemiluminescence reagents (Applygen Technologies, China). The relative levels of Stat5 phosphorylation in lymphocytes were determined by densitometry analysis.
The levels of lung anti-elastin antibodies and serum and lung iNOS were measured by ELISA using a specific kit (Ya'anda Biological Technology, Beijing, China), according to the manufacturers' protocol.

Histology was performed as previously described [20 (link)]. Briefly, freshly isolated pancreata were fixed in 4% PFA, embedded in paraffin, and sectioned at 5 μm onto charged slides. Slides were rehydrated using toluene and decreasing concentrations of ethanol. We used a microwave and citrate buffer method of antigen unmasking, TBS + 0.4% Triton for permeabilization for 30 min, and blocked non-specific staining using TBS + 2%BSA + 0.2% Tween for 1 h. All primary and secondary antibodies were diluted in blocking buffer. Slides were incubated with primary antibody overnight at 4 °C, and all slides were incubated with secondary antibodies for 30 min at room temperature.
Antibodies used: Insulin (DAKO, #A0564, Santa Clara, CA, USA), PRLR (Bioss, # bs-6445R, Woburn, MA, USA), E-cadherin (Cell Signaling Technology, #24E10, Danvers, MA, USA), STAT5A (Abcam, #ab7969, Cambridge, UK).

JXT was purchased from Beijing Lvye Pharmaceutical Co. Ltd. (Beijing, China), Fujian (China) origin. JXT was cut into small pieces and soaked in 75% ethanol at 8 times volume overnight and then extracted 3 times with 1 h for each time. Finally, the filtrates were mixed and concentrated by a rotary evaporator with bath temperature lower than 40°C. Ethanol extract was lyophilized to powder.
The reagents and kits for determining enzyme activity were purchased from Nanjing Jiancheng Bioengineering Institute (NJBI, Nanjing, China). The antibodies for evaluating G-CSFR, Bcl-2, JAK2, pJAK2, STAT5a, pSTAT5a, STAT5b and pSTAT5b were purchased from Abcam (Abcam, USA). Amifostine was purchased from Dalian Merro Pharmaceutical Factory (Dalian, China); RPMI-1640 culture medium and fetal bovine serum (FBS) were purchased from HyClone (HyClone, USA). G-CSF ELISA kit was purchased from Shanghai Tongwei Biotech Co. Ltd. (Shanghai, China).