Hrp conjugated affinipure goat anti rabbit igg h l sa00001 2

48 h after siRNA transfection, the cells were washed twice with PBS. RIPA Lysis Buffer (BL504A, Biosharp, China) supplemented with protease and phosphatase inhibitors was added to the cells and lysed on ice for 15 min. The liquid was collected and centrifuged. Protein concentration was determined using BCA protein determination kit (P0012, Beyotime, China). Total protein was transferred to PVDF membrane (Millipore, Billerica, MA) after electrophoresis in 10% or 7.5% SDS-PAGE. After blocking the nonspecific sites on the membrane with 5% sealant for 1 h at room temperature, the membrane was applied to CASP4 (1:1000, #4450, Cell Signaling Technology, USA), NLRP1(1:1000, ab36852, abcam, USA), Vinculin (1:100000, V9264-100UL, Sigma-Aldrich), GSDMD (1:2000, TA4012, Abmart China), p-ERK (1:2000, #4370, Cell Signaling Technology, USA) and incubated overnight at 4 °C. Then, the membrane was incubated with HRP-Conjugated Affinipure Goat Anti-Rabbit IgG(H+L) (SA00001-2, Proteintech) at room temperature for 1 h. The blots were finally visualized with the ECL Chemiluminescence substrate (hypersensitive) (BL523B, Biosharp, China). The film was exposed and developed by X-ray in a darkroom. The film was scanned and the strips were and quantified by Image J (1.46R). Three repeated experiments were set up in each group.

The BCL-2 protein expression level was detected in the cells using Western blotting after incubating Hela cells (2 × 10⁵) with Apt-3-F or AS1411-F (10 μM) for 48 h. Proteins were quantified, denatured, electrophoresed, and transferred onto an FVDF membrane. This membrane was blocked with 5% skimmed milk, incubated with anti-β actin (Proteintech, 66009-1, Wuhan, China) or anti-BCL-2 (Proteintech, 26593-1), and then incubated with the second antibody HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L) (SA00001-1, Proteintech) or HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L) (SA00001-2, Proteintech). Finally, the membrane was incubated with highly sensitive ECL (Enhanced Chemiluminescent, New Cell & Molecular Biotech Co., Ltd. Suzhou, China), and blots were scanned on a ChemiDoc™ XRS system (BIORAD, Hercules, CA, USA) and analyzed with Image Lab (BIORAD).

Total protein was extracted and protein concentration was detected by BCA assay. Western blot analysis was performed as previously described [39 (link)]. EFNB2 (ab69858, Abcam, Cambridge, UK), EPHB4 (ab150545, ab98933, Abcam), LDLR (ab52818, Abcam), β-catenin (ab32572, Abcam), VLDLR (ab203271, Abcam), SCARB1(ab52629, Abcam), STAT3(ab68153, Abcam), p-STAT3(ab267373, Abcam), JAK2(ab108596, Abcam), p-JAK2(ab108596, Abcam), SREBP2(ab30682, Abcam), proliferating cell nuclear antigen (PCNA; Proteintech Group, Inc., Sankt Leon-Rot, Germany) primary antibodies were used. Horseradish peroxidase (HRP)-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2) and HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1) were obtained from Proteintech Group, Inc (Chicago, US).

Total proteins from 6 day-old progeny testes, 16 week-old progeny livers and testes were extracted immediately after the mice were sacrificed. The protein concentration in the extracts was determined using a BCA protein assay kit (BL521A, Biosharp, China). Proteins were denatured, separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked in 5% non-fat milk at room temperature for 2 h. The membranes were then washed three times using Tris buffered saline tween (TBST) for 10 min each. Subsequently, the membranes were incubated with primary antibodies according to the manufacturer’s instructions, including VDAC1/2 (10866-1-AP, 1:1,000, Proteintech, China), LAMP2 (27823-1-AP, 1:1,000, Proteintech, China), and β-Actin (66009-1-Ig, 1:1,000, Proteintech, China). Next, the membranes were rinsed and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (HRP-conjugated Affinipure Goat Anti-Rabbit IgG(H + L), SA00001-2, 1:4,000; HRP-conjugated Affinipure Goat Anti-Mouse IgG(H + L), SA00001-1, 1:4,000; both from Proteintech, China). The specific immunoreactive protein bands were developed using ECL reagent (G2014-50ML, Servicebio, China).

Plasmids encoding porcine VIM (Gene ID: 100522394), TRIM21 (Gene ID: 100302538), and TUFM (Gene ID: 100516488) were constructed by inserting the synthesized sequence into pCDNA3.1 with Myc tags fused to the 3′ end and performed at Wuhan GeneCreate Biological Engineering (Wuhan, China).
The preparation work of the anti-D1133L mouse monoclonal antibody was carried out by Wuhan GeneCreate Biological Engineering (Wuhan, China). Anti-Myc rabbit monoclonal antibody (2276S), Alexa Fluor 488 anti-rabbit IgG (4416S), and Alexa Fluor 594 anti-mouse IgG (8890S) were purchased from Cell Signaling Technology (CST). HRP-conjugated goat anti-mouse IgG LCS antibody (A25012) was purchased from Abbkine and used to alleviate heavy chain interference. HRP-conjugated Affinipure Goat Anti-Mouse IgG (H&L) (SA00001-1) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H&L) (SA00001-2) were purchased from Proteintech (Wuhan, China).

PD-1 downstream signaling molecules in PBL from mice were measured by Western blot analysis at day 7 of post-infection. The primary antibodies were PI3K (ab227204, 1:1000, Abcam), p-PI3K (ab182651, 1:1000, Abcam), AKT (#9272, 1:1000, Cell Signaling Technology, USA), p-AKT (Ser473) (#9271, 1:1000, Cell Signaling Technology), ERK (#4695S, 1:1000, Cell Signaling Technology), p-ERK (Thr202/Tyr204) (#9101, 1:1000, Cell Signaling Technology), mTOR (ab2732, 1:1000, Abcam), p-mTOR (ab84400, 1:1000, Abcam), and β-actin (60008-1-lg, 1:15000, Proteintech), which was used as an internal control. The secondary antibodies were HRP-conjugated affinipure goat anti-mouse IgG (H+L) (SA00001-1, 1:8000, Proteintech) and HRP-conjugated affinipure goat anti-rabbit IgG (H+L) (SA00001-2, 1:8000, Proteintech).