β tubulin sc 55529

Rictor (sc-271081) and (β-) tubulin (sc-55529) antibodies were purchased from Santa Cruz (Santa Cruz, CA). p-Akt Ser473 (#9271) and Akt1 (#2967) antibodies were obtained from Cell Signaling Tech (Danvers, MA). Puromycin was purchased from Sigma (Shanghai, China). All the cell culture reagents were obtained from Gibco (Shanghai, China).

Normal cells HEK-293, LO2 and tumor cells B16, CT26, A549, U-2 OS, HeLa, SMMC-7721, SGC-7901, MDA-MB-231, MCF7 and MCF7/Adr were seeded in 6-well plates and grown to complete confluence. Total RNA of the cells was extracted by RNAiso Plus Reagent (Takara, Dalian, China) according to the manufacturer’s instructions. cDNA was generated from 3 μg of total RNA with High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) and the amplification of cDNA with TaqMan probes was performed using TaqMan 2* Universal PCR Master Mix (Applied Biosystems) by an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems). Primers and probes for human sstr1-5 detection in human cell lines and mouse sstr1-5 detection in mouse cell lines were designed and synthesized by GenePharma (Shanghai, China). Human and mouse gapdh genes were used as the normalization controls. The PCR process was set to run at 95 °C for 30 s, 40 cycles of 95 °C for 5 s, 55 °C for 30 s.
The protein of the above-mentioned cells was also obtained by lysing cells in cell lysis buffer (Beyotime, Shanghai, China), and separated by 10% SDS-PAGE. Western blot with antibodies sstr1 (sc-11604), sstr2 (sc-25676), sstr3 (sc-25677), sstr4 (sc-25678), sstr 5 (sc-25679), and β Tubulin (sc-55529) (Santa Cruz, CA, USA) was performed to screen the expression of sstrs in tumor and normal cells.