Anti pkcι

Immunohistochemistry (IHC) staining was performed as previously described (23 (link)). The primary antibodies included anti-PKCι (Abcam, Inc., Cambridge, UK), anti-Smoothened (Abcam, Inc., Cambridge, UK), anti-GLI1 (Abcam, Inc., Cambridge, UK), anti-HHAT (Novus Biologicals, Littleton, CO, USA), and anti-Ki-67 (ProteinTech, Chicago, IL, USA). The secondary antibodies included anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA). The proportion of PRKCI, SMO, and GLI1 staining was scored as follows: less than 1/3 = 1, between 1/3 and 2/3 = 2, or more than 2/3 = 3. The staining intensity was scored as follows: negative = 0, light yellow = 1, brownish yellow = 2, or tan = 3. The final score for PRKCI, SMO, and GLI1 expression was calculated by multiplying the 2 scores. The slides were classified into low- and high-expression groups, corresponding to scores of <6 and ≥6, respectively. The histopathological diagnosis of the patients included in our study was established by two pathologists who specialized in gynecologic oncology.

Total protein was extracted from cells using radioimmunoprecipitation assay (RIPA) lysis buffer (CW Biotechnology, Beijing, China) with protease and phosphatase inhibitors (Solarbio, Beijing, China; and CW Biotechnology, Beijing, China). According to the manufacturer’s protocol, nuclear and cytosolic lysates were extracted by using the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). Western blotting was performed as described previously (25 (link)). The following antibodies were utilized: anti-PKCι (Abcam, Inc., Cambridge, UK), anti-Smoothened (Abcam, Inc., Cambridge, UK), anti-GLI1 (Abcam, Inc., Cambridge, UK), anti-HHAT (Novus Biologicals, USA), anti-pGLI1 (Thr1074) (Affinity Biosciences, Cincinnati, OH, USA), and anti-GAPDH (Cell Signaling Technology, USA). The secondary antibodies included anti-rabbit IgG (Cell Signaling Technology, USA).