Becn1 sirnas

BECN1 siRNAs were synthesized by Thermo Fisher Scientific. The plasmids pCMV-HA-BECN1 and pCMV-HA-USP14 were constructed by ourselves. Antibody against BECN1 was ordered from OriGene (TA502643). Mouse anti-β-actin antibody was purchased from Proteintech (66009–1-lg). Mouse anti-HA monoclonal antibody was ordered from Thermo Fisher Scientific (26183). Rabbit anti-HA polyclonal antibody was ordered from proteintech (51064–2-AP). K48-linkage specific polyubiquitin antibody was purchased from Cell signaling (8081). Mouse and rabbit anti-Vimentin antibodies were ordered from abcam and proteintech (ab8978, 10366–1-AP). The cell cycle-related genes Cyclin B1, CDK1, CDC25C, PCNA were purchased from proteintech (55004–1-AP, 19532–1-AP, 16485–1-AP, 10205–2-AP). The EMT markers E-cadherin, β-catenin, Snail1, and Twist1 were purchased from proteintech (20874–1-AP, 51067–2-AP, 13099–1-AP, 25465–1-AP). Mouse and rabbit anti-USP14 antibodies were ordered from OriGene and proteintech (TA324906, 14517–1-AP). TRITC-conjugated Goat anti-Rabbit IgG and FITC-conjugated Goat anti-mouse IgG were purchased from proteintech (SA00007-2, SA00003-1).

For siRNAs knockdown, when the cell density was about 50%, we carried out the experiment according to the instructions of Pufei siRNA transfection reagent kit (Pufei, 2103–100). After 48 h, the interference effect was detected by western blot using corresponding antibody. The BECN1 siRNAs were ordered from Thermo Fisher Scientific (1,299,001).
For plasmids transfection, when the cell density was about 70–80%, we carried out the experiment according to the instructions of Pufei plasmids transfection reagent kit (Pufei, 2102–100). After 48 h, the expression effect was detected by western blot using corresponding antibody.