Skimmed milk powder

Western blot analysis was performed as previously described.²⁵, ²⁶ Tissues or cells were lysed with SDS lysis buffer containing 2% SDS and 62.5 mM Tris–HCl (pH = 6.8) and incubated at 100°C for 10 min. The protein concentration was quantified using a bicinchoninic acid (BCA) quantification kit. The protein lysates were subjected to SDS‐PAGE and subsequently transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore). After blocking in 5% skimmed milk powder (BBI Life Sciences) in 1× TBST (Tris‐buffered saline with 0.1% Tween 20), the membranes were incubated with the primary antibodies overnight at 4°C and with the HRP‐conjugated secondary antibodies (ZSGB‐BIO) for 1 h at RT, followed by visualization using the ChemiDoc XRS system (Bio‐Rad). The band intensity quantification was performed with ImageJ software. The antibodies used are listed in Table S3.

Wolfberry was purchased from Ningxia Red Goji Berry Industry Group Co., Ltd. (Ningxia, China). Bovine serum albumin (BSA), skimmed milk powder (SMP), and ovalbumin (OVA) were obtained from BBI Life Sciences Co., Ltd. (Shanghai, China). 2-Chloro-1,4-benzoquinone was purchased from Merck Life Sciences Co., Ltd. (Shanghai, China). IgG-HRP was supplied by Jackson Immuno Research Laboratories (West Grove, PA, USA). Glucose, polyethylene glycol (PEG 2000), OPD, and DAP were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). MG, LMG, crystal violet (CV), leucocrystal violet (LCV), methylene blue (MB), azure B (AZB), and azure C (AZC) were provided by Dr. Ehrenstorfer GmbH (Augsburg, Germany). White opaque microplates were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). MG-OVA and MG-Ab were purchased from Pharmaceutical Nest Bioengineering Co., Ltd. (Shanghai, China). All chemicals are of analytical grade. Deionized water with a resistance of >18.2 MΩ·cm⁻¹ was used in the experiment.

Western blot was performed as previously described (Wang et al., 2022 (link)). Tissues or cells were lysed with SDS lysis buffer [containing 4% SDS and 100 mmol/L Tris-HCl (pH = 6.8)] and incubated at 100°C for 10 min. To detect phosphorylated protein, RIPA lysate buffer (Beyotime, P0013B) containing a phosphatase inhibitor (Roche,4906837001) was used. A BCA Kit was used to perform protein quantification. The protein lysates were subjected to SDS-PAGE and subsequently transferred to a PVDF (polyvinylidene fluoride) membrane (Millipore). The membranes were blocked in 5% skimmed milk powder (BBI Life Sciences) in 1× TBST, incubated with the primary antibodies overnight at 4°C and with the HRP-conjugated secondary antibodies for 1 h at RT (ZSGB-BIO), followed by visualization using the ChemiDoc XRS system (Bio-Rad). The quantification was performed with Image J software. The antibodies used are listed in Table S5.