Hrp conjugated affinipure goat anti mouse igg h l sa00001 1

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).

The Wild (WT) and Mutant (MT) CLTC sequence of exon 19 to exon 23 (E19-E23) and exon 19 to exon 23 (E19-E21) with an N-terminal eGFP tag was cloned to the expression vectors. The plasmids were transfected to the HEK293t cells. The primary antibodies GFP Monoclonal antibody (66002-1-Ig, Proteintech Group) and GAPDH Monoclonal antibody (60004-1-Ig, Proteintech Group) were used. The second antibody used in the study was HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1, Proteintech Group).

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).

Cells were lysed in RIPA buffer (P0013B, Beyotime) with protease inhibitors (GRF101, Epizyme, China). After centrifugation at 14000 × g, 4 °C for 15 min, proteins were denatured using 5 × loading buffer (G2013-1ML, Servicebio) and boiled for 10 min. Equal quantities of total protein per lane were separated by SDS-PAGE and transferred to PVDF membranes (IPVH00010, Merck Millipore). The membranes were washed in 5% non-fat dry milk in TBST for 1 h, and were then incubated at 4 °C overnight with a different primary antibody. Next day, membranes were incubated with the corresponding secondary antibodies for 1 h at room temperature, and visualized with Enhanced Chemiluminescent (ECL, New Cell & Molecular Biotech, China) by using ChemiDoc Imaging Systems (Bio-Rad).
The primary antibodies were listed as following: GAPDH (60,004–1-Ig, Proteintech), β-actin (66009–1-Ig, Proteintech), Nrf2 (16396–1-AP, Proteintech), SND1 (10760–1-AP, Proteintech), METTL3 (15073–1-AP, Proteintech), METTL14 (26158–1-AP, Proteintech), WTAP (10200–1-AP, Proteintech), FLAG (20543–1-AP, Proteintech), Keap1 (10503–2-AP, Proteintech), GPX4 (67763–1-Ig, Proteintech), 4-Hydroxynonenal (4-HNE, R&D systems, MAB3249). Secondary antibodies used were HRP-conjugated Affinipure Goat Anti-Mouse IgG (H + L) (SA00001-1, Proteintech) and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00001-2, Proteintech).