Mouse anti gapdh mab

For the GFFs, 1 × 10⁶ positive GFFs and WT GFFs were transferred into a 60 mm culture dish, then 10 µg/mL Brefeldin A (Beyotime Biotechnology) was added for 6 h. The cells were resuspended in PBS and centrifuged again. Then, the cells or tissues were lysed in RIPA lysis buffer (Solarbio, Tongzhou, Beijing, China) containing protease inhibitor cocktails (Beyotime Biotechnology). Briefly, the lysates were incubated on ice for 15 min, then centrifuged at 14,000 rpm for 10 min at 4 °C. Protein concentrations were determined by a BCA Protein Assay Kit (CWBIO) assay. Samples were separated by electrophoresis on SDS-PAGE gels and transferred onto polyvinylidene fluoride membranes (Solarbio). The samples were blocked for 120 min in Western Blocking Buffer (Solarbio). Then, the PVDF membranes were incubated overnight with mouse anti-GAPDH mAb (ZSGB-BIO) and BChE polyclonal antibody (Proteintech 23854-1-AP) (1:500). After being washed three times with TBST, PVDF membranes were incubated with horseradish-specific rabbit anti-goat IgG (H +L) (ZSGB-BIO) and horseradish-specific mouse anti-goat IgG (H +L) (ZSGB-BIO) for 60 min. Protein bands were visualized using the Bioanalytical Imaging System (Azure Biosystems, Dublin, CA, USA).

ND‐1, the mAb of LEA, was obtained and purified as described previously.16 Mouse anti‐PODXL mAb (3D3, cat#sc‐23904) and normal mouse IgG (cat#sc‐2025) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti‐TSG101 mAb (cat#ab30871) was obtained from Abcam (Cambridge Science Park, Milton, Cambridge, UK). Mouse anti‐CD63 mAb (cat#10628D) was purchased from Invitrogen Corporation (Carlsbad, CA, USA). Mouse anti‐actin mAb (TA‐09), mouse anti‐GAPDH mAb (TA‐08), sheep anti‐mouse FITC‐IgG (ZF‐0315), sheep anti‐mouse IgG‐HRP (ZDR‐5307), and sheep anti‐rabbit IgG‐HRP (ZDR‐5306) were obtained from ZSGB‐BIO (Beijing, China). The plasmid pcDNA3.1‐PODXL was a generous gift from Dr. Ayuso MS, CSIC, Madrid, Spain.

Cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (C1053, Beijing Applygen Technologies Inc.), and protein concentrations were determined using the bicinchoninic acid (BCA) Protein Assay Kit (PC0020, Solarbio, China). Total protein of 20 μg was separated on a 10% NuPAGE^TM Bis-Tris Welcome Pack (NP030B, Invitrogen), and the electrophoresed products were transferred to iBlot^TM 2 NC Regular Stacks (IB23001, Life Technologies). Membranes were blocked for 30 min at RT using 5% powdered milk, and primary antibodies mouse anti-eGFP mAb (TA-06, ZSGB-BIO, China) and mouse anti-GAPDH mAb (TA-08, ZSGB-BIO, China) were incubated overnight at 4°C. After washing the membranes, the secondary antibody Goat anti-Mouse IgG (H + L) (ZB-2305, ZSGB-BIO, China) was incubated for 2 h at RT. Bands were visualized with Pro-light HRP substrate chemiluminescent system (PA112, Tiangen Biotech Co., Ltd.). Chemiluminescent signals were quantified using ImageJ software.