Rabbit anti cd133

The cultured cells were washed with cold PBS and then treated with cell lysis buffer (2× loading buffer, 2 μg·mL⁻¹ Aprotinin, 1 mm PMSF, 2 mm β‐mercaptoethanol) at 100 °C for 10 min. Then the mixture was centrifuged under 4 °C at 14000g for 10 min. Approximately 10 μL of protein was loaded in each lane of 10% SDS/PAGE gel and separated, and the protein then transferred to a PVDF membrane. The membrane was blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies at 4 °C overnight, followed by the secondary antibody. The antibodies used were rabbit anti‐CD133 (Cell Signaling Technology, Danvers, MA, USA; #64326), rabbit anti‐OCT4 (Cell Signaling Technology; #2750), rabbit anti‐SOX2 (Cell Signaling Technology; #3579), rabbit anti‐NANOG (Cell Signaling Technology; #4903), mouse anti‐KRAS (Santa Cruz Biotechnology, Dallas, TX, USA; sc30), mouse anti‐β‐Tubulin (Cell Signaling Technology; #6181), rabbit anti‐TET3 (Cell Signaling Technology; #85016), rabbit anti‐Lin28B (Signalway Antibody LLC, College Park, MD, USA；#21626), rabbit anti‐PKCβ (Cell Signaling Technology; #46809), rabbit anti‐PKCδ (Cell Signaling Technology; #2058), rabbit anti‐PKCζ (Cell Signaling Technology; #9372), rabbit anti‐PKCμ (Cell Signaling Technology; #2056) and mouse anti‐Flag (Sigma; CAT F1804).

PFK118–310 was purchased from Sigma-Aldrich while ICG-001 was obtained from Selleck Chemicals. Alexa fluor 488 phalloidine and 4′, 6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. The antibodies used for Western blot analysis and immunocytochemistry were: rabbit anti-CD44 (Abcam # 51037), rabbit anti-CD133 (Cell Signaling # 3663), mouse anti-CD166 (Abcam # 175422), rabbit anti-Lgr5 (Sigma-Aldrich # HPA012530), goat anti-actin (Santa Cruz # sc-1615), rabbit anti-beta-catenin (Cell Signaling # 8480), mouse antibodies specific for the active form of beta-catenin, dephosphorylated on Ser 37 and Thr 41 (Millipore, clone 8E7, # 05–665). Anti-rabbit IgG horseradish peroxidase-linked antibodies and anti-mouse IgG horseradish peroxidase-linked antibodies were from Cell Signaling whereas Cy3-conjugated anti-mouse secondary antibodies were obtained from Jackson ImmunoResearch Labs.
The following antibodies were used for flow cytometry analysis: phycoerythricine-conjugated mouse anti-human ABCB1 (BD Biosciences # 557003), phycoerythricine-conjugated mouse anti-human ABCG2 (BD Biosciences # 561180), mouse anti-human ABCC1 (BD Biosciences # 557594) and anti-mouse phycoerythricine-conjugated secondary antibodies (BD Biosciences # 555574).