Complete protease inhibitor and phosphatase inhibitor cocktail

BMDMs and THP1 cells were lysed in ice-cold RIPA buffer supplemented with complete protease inhibitor and phosphatase inhibitor cocktails (Roche). Protein concentration was measured by the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific-23227). Serum was isolated from mouse blood by centrifugation at 10,000 RPM for 10 min at 4°C. The concentration of IL-6, IL-1β, and TNF-α in cell culture medium and serum was measured using commercially available ELISA kits (R&D Systems). SARS-CoV-2 S protein in cell lysates was detected by SARS-CoV-2 S ELISA Kit and following the manufacturer’s instruction (RayBiotech, ELV-COVID19S2).

Skin tissue and cell samples were homogenized in RIPA lysis buffer (50 mM Tris at pH 7.4, 150 mM NaCl, 0.5% Sodium deoxycholate, 0.1% SDS and 1% Triton X-100 with complete protease inhibitor and phosphatase inhibitor cocktails [Roche]) and then denatured in SDS sample buffer (50 mM Tris at pH 6.8, 100 mM dithiothreitol, 2% SDS and 10% glycerol) for 10 min at 100 °C. The extracted proteins were separated by SDS–polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA), followed by electro-transferred to a polyvinylidene fluoride membrane (PerkinElmer, Waltham, MA, USA). The membranes were blocked with 5% (w/v) non-fat dried milk solution, then incubated with the required specific antibody. This was followed by detection by visualizer kit (Millipore, Burlington, MA, USA, WBKLS0500). The following antibodies were used for the Western blotting: Cisd2 [29 (link)], Gapdh (Millipore, MAB374), β-tubulin (Millipore, 05661) and MMP-1 (Proteintech, Rosemont, IL, USA, 10371-2-AP).

THP1 cells, BMDM, HEK293T, A549, and Calu3 cells were lysed in ice-cold RIPA lysis buffer containing complete protease inhibitor and phosphatase inhibitor cocktails (Roche), resolved by SDS-PAGE, and transferred onto a PVDF membrane. The membranes were immunoblotted with antibodies against Phospho-NF-κB p65 (3033, Cell Signaling), Phospho-IκBα (9246, Cell Signaling), IκBα (4812, Cell Signaling), Phospho-ERK (4370, Cell Signaling), ERK (4695, Cell Signaling), Phospho-JNK (4668, Cell Signaling), Phospho-AKT (4060, Cell Signaling), Phospho-STAT3 (9145, Cell Signaling), SARS-CoV-2 S (GTX632604, GeneTex), and β-actin (A2228, Sigma-Aldrich). Immunoreactive protein bands were detected using ECL super signal west femto substrate reagent (Thermo Fisher Scientific).

For immunohistochemical analysis, postmortem human brain tissue was obtained through the brain donation program of the Morris K. Udall Parkinson's Disease Research Center of Excellence and the Alzheimer's Disease Research Center at Johns Hopkins Medical Institutions (JHMI) in compliance with local Institutional Review Board and HIPAA regulations. Formalin-fixed, paraffin-embedded tissue sections (10-µm thick) containing cingulate cortex tissue from two normal control and four PD/DLB brains was obtained as indicated in Table 1 and described previously (34 (link)). For biochemical analysis, caudate putamen tissue derived from five normal control and seven PD/DLB subjects was obtained from JHMI (Table 1), and frontal cortex tissue derived from four normal control and eight PD subjects was obtained from the archive at Queen Square Brain Bank (QSBB) as previously reported (35 (link)) and indicated in Table 2. Flash-frozen tissues were homogenized with (10% w/v) lysis buffer [20 mM Tris–HCl pH 7.4, 150 mM NaCl, Complete protease inhibitor and phosphatase inhibitor cocktails (Roche Applied Science)] and equal proteins were resolved by 3–8% Tris–acetate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen). Blots were probed with anti-VPS35, anti-actin or anti-β-tubulin antibodies.

Liver tissues from each mouse from the three different groups were homogenized in lysis buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1 mM EDTA, and 1% Triton X‐100 in the presence of complete protease inhibitor and phosphatase inhibitor cocktails (Roche). The hepatic lysates (10 μg proteins) were separated by 12% SDS‐PAGE and transferred onto polyvinylidene fluoride (PVDF) membrane. The membranes were blocked using 5% (w/v) non‐fat freeze‐dried milk in buffer, then probed with anti‐Cisd2 (Chen et al., 2009a (link)) or anti‐Gapdh (MAB374, Millipore) antibody, and finally developed using ECL reagents (34580, Thermo). Quantitative densitometric analysis using Image J software was performed.

Eca 109, Eca 9706, and KYSE-510 cells were starved in serum-free medium overnight, and SHR6390 were added for 24 h. The Cell pellets and tumor tissues of xenografts were lysed using RIPA Lysis Buffer (Beyotime Biotechnology, Jiangsu, China) on ice, containing complete protease inhibitor and phosphatase inhibitor cocktail (Roche, Switzerland). Protein concentrations were measured using the BCA Protein Assay Kit (Beyotime Biotechnology, Jiangsu, China). Protein samples were diluted to equal concentrations (40 μg), and separated by electrophoresis in 10–12% SDS-PAGE and transferred onto nitrocellulose membranes (GE Healthcare, Piscataway, NJ). Antibodies used were against: CDK6 (sc-177) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); Rb(#9313S), pRb(#9307S), CDK4(#12790), Cyclin D1 (#2978) (Cell Signaling Technology, Boston, MA, USA); β-actin (#014M4759) (Sigma-Aldrich, USA). All antibody except CDK6 (1:100) dilutions were 1:1000. Proteins were visualized using ECL plus Western Blotting Detection Reagents (GE Healthcare). Densitometry analysis of the Western blot protein was performed using the ImageJ software.