Iblot 2 pvdf regular stacks

Cells used for Western blotting were stimulated in FBS-free media. Cells were lysed in RIPA lysis buffer (Merck Millipore, Burlington, MA), and the protein concentrations were measured using the Micro BCA™ Protein Assay (Thermo Scientific). The proteins were separated in 8-16% stain-free TGX gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes (iBlot® 2 PVDF regular stacks, Invitrogen, Carlsbad, CA), and analysed by immunoblotting. The primary antibodies used were cleaved IL-1β (1 : 1000 Cell Signaling Technologies, Danvers, MA. cat.nr:12242), IL-18 (1 : 1000 Abcam, Cambridge, UK. cat.nr: EPR19954-188), and caspase-1 p20 (1 : 750 Adipogen Life Sciences, cat.nr:AG-20B-0048-C100). Secondary antibodies used were goat anti mouse (1 : 5000 Abcam cat.nr: Ab6789), rabbit anti goat (1 : 2000 Dako, Agilent. Santa Clara, CA. cat.nr: P0160), and goat anti-rabbit (1 : 3000 Invitrogen cat.nr: A11034). Membranes were washed in TBST buffer and analysed with ChemiDoc™ MP Imaging System (Bio-Rad).

Freshly harvested cells were lysed in RIPA buffer. Protein concentrations were determined by Pierce BCA^™ Protein Assay Kit (Pierce). Proteins were separated via Mini Protean TGX stain free gel 4–15% (BioRad) and transferred to polyvinilydene difluoride membrane by using iBlot 2 PVDF Regular Stacks (Invitrogene) and iBlot system transfer system (LifeTechnologies).
Membranes were blocked in 5% BSA solution (Sigma). Primary antibodies were diluted following the manufacturer’s instructions: anti-Vinculin antibody (Cell Signaling) (1:1000) and antiCHD1 (Novus Biologicals) (1:2000).
Signals were developed by using Clarity Western ECL Substrate (BioRad) and Image Quant LAS4000 System (GEHealthCare).

To investigate the reaction specificities of pp38 mAbs to distinct serotypes of MDV, the CEF monolayers were separately infected with Md5, GX0101, GA, CVI988, HVT, and SB-1 viruses to produce MDV plaques, and then the CEF cells were fixed and incubated with the newly identified pp38 mAb 31G7 to perform IFA staining. Simultaneously, the virus-infected CEF cells were collected and boiled with 1×SDS–PAGE Sample Loading Buffer (Beyotime, China) for 10 min. The samples were separated on Bolt^TM Bis-Tris Plus 4%–12% precast gel (Invitrogen), and the resolved proteins were then transferred onto PVDF membranes by iBlot^® 2 PVDF Regular Stacks (Invitrogen, USA). The expression levels of pp38 proteins in Md5, GX0101, GA, CVI988, HVT, and SB-1-infected CEFs were determined by incubating mAb 31G7 and HRP-labeled Goat Anti-Mouse IgG (Pharmacia, USA) sequentially, and were finally visualized using NcmECL Ultra (NCM Biotech, China). In all cases, chicken β-actin (Sangon Biotech, Shanghai, China) served as the loading control.

Cells were seeded in 6-well plates and treated with SFM. Thereafter, the cells were treated with uRO-w, EA, UA, or DEX and cultured for 24 h. After removing the supernatant, the cells were washed and treated with 0.5 ng/mL IL-1β for 30 min. For protein extraction, the cell pellet was lysed using Tris-glycine SDS-sample buffer (2×) and electrophoresed on a 10% Tris-glycine gel. Subsequently, the membranes were transferred to iBlot^® 2 PVDF Regular Stacks (Invitrogen, Carlsbad, CA, USA) and blocked for 1 h using a blocking buffer. Next, the membrane was incubated with primary antibodies against AKT, phospho-AKT, p38, phospho-p38, ERK, phospho-ERK, JNK, phospho-JNK, NF-κB, phospho-NF-κB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at 4 °C overnight. After washing, the membranes were further incubated with an appropriate dilution of secondary antibody. The blot was developed using an ECL substrate, and images were analyzed using the iBright™ FL1500 Imaging System (Thermo Scientific, Madison, WI, USA). The antibodies used for protein detection are listed in Table 1.

To investigate the reaction specificities of newly developed Meq mAbs to different virulence of MDVs and HVT, the CEF monolayers were infected with GX0101, Md5, GA, CVI988, SB-1 or HVT viruses separately to produce virus plaques, and the cells were fixed and incubated with ascitic fluids of the newly identified Meq mAbs, diluted in 1:5000 or 1:2000 for 2A9-B11 and 8G11-B2, respectively, to perform IFA staining. Simultaneously, the virus-infected CEF cells were collected and boiled with 1 × SDS-PAGE Sample Loading Buffer (Beyotime) for 10 min. The samples were separated on Bolt^TM Bis-Tris Plus 4–12% precast gel (Invitrogen), and the resolved proteins were then transferred onto PVDF membranes by iBlot^® 2 PVDF Regular Stacks (Invitrogen). The expression levels of Meq in each virus-infected CEFs were determined by incubation of newly identified Meq mAb and HRP-labeled Goat Anti-Mouse IgG (Pharmacia) sequentially and were finally visualized using NcmECL Ultra (NCM). In all cases, the chicken β-actin (Sangon Biotech, Shanghai, China) serves as the loading control.

Protein samples were run on NuPAGE 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) at 100 V and transferred in the iBlot 2 system (Invitrogen) to polyvinylidene difluoride (PVDF) membranes (iBlot 2 PVDF regular stacks; IB24001). Membranes were fixed for 20 min in 4% paraformaldehyde (PFA) in PBS. PVDF membranes were blocked in the Odyssey blocking buffer, PBS (LI-COR, ref. 927-40000) for 1 h. Membranes were incubated for 1 h at room temperature (RT) or overnight at 4 °C with primary antibodies in Odyssey blocking buffer, PBS, 0.2% Tween-20 (PBS-T), and washed 3 × 10 min at RT in PBS-T. Membranes were incubated protected from light for 1 h at RT in secondary antibodies in the same buffer and washed 3 × 10 min at RT in PBS-T. Membranes were then scanned on Odyssey CLx (LI-COR).

Samples were electrophoresed on NuPAGE 4–12% Bis–Tris gels with NuPAGE MES-SDS running buffer and SeeBlue Plus2 molecular weight marker (all by Invitrogen) at 120 V and transferred in the iBlot 2 system (Invitrogen) to PVDF membranes (iBlot 2 PVDF regular stacks; IB401031). Membranes were fixed for 10 min in 4% paraformaldehyde (in PBS). Membranes were blocked in blocking buffer (TBS blocking buffer, LiCor 927–60,001) for 1 h and incubated in primary antibody in TBST Licor blocking buffer overnight at 4 °C. Membranes were washed 3 × 10 min in TBST. Secondary antibodies were prepared in the blocking buffer and incubated for 1 h at RT. Membranes were washed 3 × 10 min in TBST and scanned (Odyssey CLx, Li-Cor).