Anti gsk3β

Antibodies used in this study include Anti-SGK196 (ab57908, Abcam, Cambridge, UK); Anti-GSK3β (A0480, Abclonal, Wuhan, China), Anti-p-GSK3β-S9 (AP0039, Abclonal); Anti-RPN1 (sc48367, Santa Cruz, Dallas, TX); Anti-p-AKT-Ser473 (#4058, Cell Signaling Technology, Danvers, MA), Anti-p-AKT-Thr308 (#13038, Cell Signaling Technology), Anti-AKT(pan) (#2920, Cell Signaling Technology), Anti-Snail (#3879, Cell Signaling Technology); Anti-HA (H6908, Sigma-ALDRICH, MO), Anti-Flag (F1804, Sigma-ALDRICH), Vimentin (sc-66001,Santa Cruz), α-dystroglycan (VIA4) (sc-53986, Santa Cruz) and Anti-GAPDH (HC301-01, TRANSGEN BIOTECH, Beijing, Shanghai). All antibodies for Western blotting were used at dilution 1:1000. For Immunofluorescence assays, Anti-HA (H6908, Sigma-ALDRICH) was used at dilution 1:100. Other reagents include DMEM medium (11965-084, Gibco), RPMI 1640 medium (11875-085, Gibco), Cell Counting Kit reagents (40203ES60, YEASEN), puromycin (P8230, Solarbio, Beijing, China), LY294002 (L9908, Sigma-ALDRICH), CHX (5087390001, Sigma-ALDRICH), Protease inhibitor Cocktail (EDTA-Free, 100x in DMSO, 20124ES03, YEASEN), Endo H (# P0702S, New England Biolabs, Ipswich, MA), and PNGase F (# P0705S, New England Biolabs).

Total proteins were extracted using RIPA lysis buffer and protein concentration was measured using a BCA assay kit (Beyotime). About 30 μg proteins were separated by SDS‐PAGE and then transferred onto nitrocellulose membranes. After blocking with 5% non‐fat milk, the membranes were incubated sequentially with primary antibodies and horseradish peroxidase (HRP)‐conjugated secondary antibodies. The primary antibodies used included antibodies by Abcam: anti‐low‐density lipoprotein receptor‐related protein 6 (LRP6) (ab134146), anti‐ALP (ab108337), and anti‐osterix (ab94744); antibodies by Cell Signalling Technology: anti‐PPARγ (#2443), anti‐C/EBPα (#8178); anti‐Runx2 (#12556), anti‐phospho‐LRP6 (S1490) (#2568), anti‐non‐phospho‐β‐catenin (#8814), anti‐phospho‐GSK3β (S9) (#5558), anti‐phospho‐Smad1/5(S463/465) (#9516) and anti‐GSK3β (#12456); antibodies by Abclonal: anti‐Smad5 (A1947); antibodies by Proteintech: anti‐Smad1 (10429‐1‐AP); anti‐β‐catenin (51067‐2‐AP), anti‐osteopontin (25715‐1‐AP); anti‐fatty acid binding protein 4 (FABP4) (12802‐1‐AP), and anti‐β‐actin (66009‐1‐Ig). The bands were visualized by chemiluminescence reagent (Proteintech). The relative expression levels of the target genes were calculated by dividing the intensity of the target genes by that of β‐actin.

The reagents used were as follows: sodium new houttuyfonate (Yuanye, Shanghai, China, CAS: 112714-99-5); docetaxel (Yuanye, Shanghai, China); hydroxypropyl-β-cyclodextrin (HP-β-CD; Solarbio, Beijing, China); N-acetyl-cysteine (NAC; Macklin, Shanghai, China); matrix adhesive (Biozellen, Frontier, NE, USA); Opti-MEM I medium (Gibco, Billings, MA, USA); and crystal violet (BioSharp, Hefei, China).
The kits used were as follows: Cell Counting Kit-8 (Hycezmbio, Wuhan, China), ROS Detection Kit (Hycezmbio, Wuhan, China), BCA Protein Quantification Kit (Hycezmbio, Wuhan, China), Apoptosis Detection Kit (Hycezmbio, Wuhan, China); and Transwell chamber (Corning, NY, USA).
According to the protocol recommended by the manufacturer, the following antibodies were used for Western blot or immunofluorescence: Anti-BAX (Wanleibio, WL01637), Anti-p-GSK3β (Wanleibio, WL03683), Anti-β-actin (ABclonal, AC038), Anti-Bcl-2 (ABclonal, A19693), Anti-cleaved PARP p25 (ABclonal, A19612), Anti-caspase-9 (ABclonal, A0281), Anti-PDK1 (ABclonal, A0834), Anti-p-PDK1 (ABclonal, AP0426), Anti-AKT (ABclonal, A20799), Anti-p-AKT (ABclonal, WLP001), Anti-GSK3β (ABclonal, A11731), Anti-MMP1 (ABclonal, A22080), HRP Goat Anti-Rabbit IgG (ABclonal, AS014), Alexa Flour 594-Goat Anti-Rabbit IgG (ABbox, AD9279), and Cy3 Goat Anti-Rabbit IgG (H + L) (ABclonal, AS007).