cDNA was prepared according to standard methods: RNA was reverse-transcribed with oligo(dT) primer using 1 μg total RNA in a total volume of 20 μl containing transcription buffer, RNase Inhibitor, Prime Script™ RTase. For PCR, 30 cycles of denaturation (94°C for 45s), annealing (60°C for 45s), and elongation (72°C for 1 min) was performed using the following primer pairs for HIF-1α : BLZ945 clinical trial forward: 5′-TGGACTCTGATCATCTGACC-3′, reverse: 5′-CTCAAGTTGCTGGTCATCAG-3′, which yielded a 434-bp product. 30 cycles of denaturation (95°C for 1 min), annealing (55°C for 60s), and elongation (72°C for 1 min) were performed using the following primer pairs for
MDR1 : forward: 5′-GAATCTGGAGGAAGACATGACC-3′, reverse:5′-TCCAATTTTGTCACCAATTCC-3′, which yielded a 259-bp product.35 cycles of denaturation
selleck products (95°C for 30s), annealing (50°C for 1 min), and elongation (72°C for 1 min) were performed using the following primer pairs for MRP1 : forward: 5′-TCAGCCCTTCCTGACAAGCT-3′, reverse: 5′-TCTCTGCTGCAGGAGGTCCG-3′, which yielded a 318-bp product. The GAPDH  control PCR was performed using the following primer pairs: forward: 5′-ACCACCATGGAGAAGGCTGG-3′, reverse: JQEZ5 5′-CTCAGTGTAGCCCAGGATGC-3′, which yielded a 527-bp product. For negative controls, the PCR reaction was performed without prior reverse transcription. Amplified cDNA was visualized by ethidium bromide staining on 1.5% agarose gels on a Bio-Rad gel scanner (Bio-Rad, USA). Western Blot The chordoma cell line CM-319 and frozen nucleus pulposus tissues were harvested and lysed with a cold RIPA protein lysis buffer for 30 minutes on ice. The lysates were transferred to Eppendorf tubes and clarified by centrifugation at 12,000 g for 10 minutes at 4°C. The supernatant was kept in -80°C for future use. The BCA method was performed to determine
the protein concentration in the supernatant. Samples (30 μg of total protein each) were boiled at 95°C for five minutes and loaded onto SDS-PAGE (5% stacking gel and 8% separating gel), followed with a separation at 80 volts for about two hours and subsequent transferred onto a nitrocellulose membrane. The membrane was blocked in 5% defatted milk for 1 hour at room temperature, and was then incubated in the primary antibodies diluted in 5% defatted milk/TBST overnight at 4°C (MDR1 1:200, mouse Thiamet G anti-human, Santa Cruz; MRP1, 1:200, rabbit anti-human, Santa Cruz; HIF-1α, 1:200, rabbit anti-human, Santa Cruz). The membrane was washed three times with TBST and incubated with the second antibodies for an hour at room temperature, then washed three times with TBST again. The enhanced chemiluminescene (ECL) system (Piece) was used for detection of MDR1, HIF-1α and MRP1. Protein bands were visualized and quantified using Quantity-One software (Bio-Rad USA). The MDR1, HIF-1α and MRP1 bands were visualized at an apparent molecular weight of 170, 120 and 190 kDa, respectively.
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