Goat anti rabbit igg h l hrp

BMDMs were lysed with 1.5x Laemmli’s Sample Buffer in H₂O and incubated at 95°C for 3 min. Cell lysates were size-fractioned by 8% SDS-PAGE and wet-transferred to a nitrocellulose membrane. Membranes were blocked with 5% non-fat dry milk in Tris buffered saline with 0.1% Tween-20 (TBST) at room temperature. Western blotting was performed using the following antibodies: anti-NLRX1 (1/1000, Proteintech, 17215-1-AP), anti-y-TUBULIN (1/10 000, Sigma-Aldrich, T5326), goat anti-rabbit IgG (H+L) HRP (1/2500, Promega, W4011) and goat anti-mouse IgG (H+L) HRP (1/2500, Promega, W4021). ECL Western Blotting detection reagent (GE Healthcare Life Sciences) was used for revelation.

SG tissues were ground to extract total protein. The total protein concentration was determined using the BCA Protein Assay Kit (Cwbio, China). Equal amounts of proteins were subjected to 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Membranes were blocked using 5% skimmed milk and subsequently incubated overnight at 4°C with anti-calmodulin (CALM) (Bioss Antibodies, China; 1:1,000), anti-tropomyosin 1 (TPM1) (Boster, China; 1:1,000), anti-tropomyosin 2 (TPM2) (Bioss Antibodies, China; 1:1,000), and anti-β-actin (TransGen Biotech, China; 1:5,000). Then, the membranes were incubated using secondary antibodies, including goat anti-Mouse IgG (H+L) HRP (Promega Corporation, USA; 1:5,000), or goat anti-rabbit IgG (H+L) HRP (Promega Corporation, USA; 1:5,000). Last, protein signals were assessed and analyzed using the ECL western blot detection system (Millipore, Billerica, MA, USA).

The following antibodies were used for immunoblotting: Rat anti-HA (Roche, ROAHAHA), Rabbit anti- FLAG (Proteintech, 20543-1-AP), Mouse anti-GAPDH (Developmental Studies Hybridoma Bank, hGAPDH-2G7), Rabbit anti-USP46 (Proteintech, 13502-1-AP), Mouse anti-Tubulin (Developmental Studies Hybridoma Bank, E7), Mouse anti-β-catenin (BD Transduction Laboratory, 610154), Rabbit anti-Axin1 (Cell Signaling Technology, 2087), Rabbit anti-Insulin Receptor β (Cell Signaling Technology, 3025), Rabbit anti-LRP6 (Cell Signaling Technology, 2560), Rabbit anti-WDR20 (Bethyl Laboratories, A301-657A), Rabbit anti-WDR48 (UAF1) (Proteintech, 16503-1-AP), Goat anti-rat IgG H + L-HRP (Thermo, 31470), Goat anti-mouse IgG H + L-HRP (Promega, W4021), Goat anti-rabbit IgG H + L-HRP (Promega, W4011). All primary antibodies were used at 1:1000 dilution except anti-FLAG (1:2000), anti-GAPDH (1:500), and anti-WDR20 (1:2500). All secondary HRP antibodies were used at 1:5000 dilution.