Hrp conjugated goat anti rabbit or anti mouse secondary antibodies

Cells were lysed in RIPA buffer containing proteinase inhibitors. Equal amount of proteins was separated by a 12% SDS-PAGE gel. Following electrophoresis, proteins were transferred to a PVDF membrane, blocked in 5% non-fat milk, and incubated with primary antibodies at 4 ^oC overnight. Antibodies for ERK1/2, p-ERK1/2, NF-кB p65, p-p65, STAT3, p-STAT3, p-p38, p38, p-Akt, Akt, LC3, CD9, CD63, Alix, and TSG101 were purchased from Cell Signaling Technology (Louis Park, MN, USA). After washing with TBST for three times, membrane was incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bioworld Technology) at room temperature for two hours. The protein bands were visualized by enhanced chemiluminescence. GAPDH served as the loading control.

Western blotting was performed as previously described [47 (link)]. Briefly, proteins were extracted from tissue samples or neutrophils using RIPA lysis buffer containing proteinase inhibitors. Equal amounts of protein (20 μg) were separated by 10% or 12% SDS‒PAGE. Following electrophoresis, proteins were transferred to a PVDF membrane, blocked in 5% nonfat milk, and incubated with primary antibodies at 4 ℃ overnight. Antibodies against HIF-1α and citH3 were purchased from Abcam (UK), and antibodies against ERK1/2, p-ERK1/2, p65 NF-кB, p-p65 NF-кB, p38 MAPK, p-p38 MAPK, Akt, p-Akt, STAT3, p-STAT3 and GAPDH were purchased from Affinity Technology (USA). After washing with TBST three times, the membrane was incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bioworld Technology) at RT for 1 h. The protein bands were visualized by enhanced chemiluminescence and analysed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). GAPDH served as the loading control.

The cells were lysed in RIPA buffer containing proteinase inhibitors. Equal amount of proteins was loaded and separated on a 12% SDS-PAGE gel. Following electrophoresis, the proteins were transferred to a PVDF (polyvinylidene difluoride) membrane, blocked in 5% (w/v) non-fat milk, and incubated with the primary antibodies at 4 ^oC overnight. The sources of primary antibodies were: SALL4 (954-1053; Abnova, Taipei, Taiwan); CD44 (BS6825; Bioworld technology, Louis Park, MN, USA) and GAPDH (MB001, Bioworld technology); ERK (4695S; Cell Signaling Technology, Beverly, MA, USA), p-ERK (4370S; Cell Signaling Technology), NF-κB p65 (8242S; Cell Signaling Technology), p-NF-κB p65 (3033P; Cell Signaling Technology), STAT3 (4904P; Cell Signaling Technology), p-STAT3 (9145P; Cell Signaling Technology), N-cadherin (13116S; Cell Signaling Technology), and Oct4 (2750S; Cell Signaling Technology); Sox2 (AB5603; Merck Millipore, Shanghai, China), c-Myc (10057-1-AP; Proteintech, Rosemont, IL, USA), Nanog (AF1505; Signalway Antibody, College Park, MD, USA), E-cadherin (H-108, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with TBS/T for three times, the membranes were incubated with HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies (Bioworld technology) at room temperature for 1 h. The protein bands were visualized by enhanced chemiluminescence.