Las 3000 mini system

To evaluate the expression of different AQP genes, OA chondrocytes (n=4) were cultured in 6-well plates with DMEM for 12 h, and RNA was extracted using the QIA shredder and RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. For RT-PCR, 1 µg of total RNA was reverse transcribed to first-strand complementary DNA (cDNA) using 1.25 µM oligo-dT primers in 40 µl PCR buffer II containing 2.5 mM MgC1₂, 0.5 mM dNTP mix, 0.5 U RNase inhibitor, and 1.25 U MuLV reverse transcriptase (PerkinElmer, Inc., Foster City, CA, USA) at 42°C for 60 min. The cDNA amplification was performed under the following PCR conditions: 94°C for 5 min; followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 30 sec; and a final extension at 72°C for 2 min using AmpliTaq Gold DNA Polymerase (cat. no. N8080241; Thermo Fisher Scientific, Inc., Rockford, IL, USA) and specific primers (Table I). The amplicons, along with the TrackIt 50 bp DNA ladder (cat. no. 10488-043; Thermo Fisher Scientific, Inc.), were resolved using 3% polyacrylamide gel electrophoresis, and the signals were visualized using the LAS-3000 mini system (FujiFilm, Tokyo, Japan).

A total of 3 × 10⁶ cells were treated with 3 ng/μl dsRNA and harvested for protein isolation after five days. Harvested cells were washed twice with 1x phosphate buffered saline (PBS) and lysed by resuspension in 1x PBS supplemented with 1x Proteinase Inhibititor without EDTA (Roche) and 1% Tween. Resuspended cells were frozen in liquid nitrogen, incubated at room temperature until almost complete thawing and then transferred to ice for 30 min. After a centrifugation step (16000 x g, 4°C, 15 min) supernatant was mixed with SDS-loading buffer and comparable amounts of protein were applied to a 12% SDS-gel.
For fly extraction individual flies were anesthetized, ground in 75 μl lysis buffer (30 mM HEPES, 100 mM KOAc, 2 mM MgOAc, 1 mM DTT, 1% triton-X and 1x proteinase inhibitor cocktail (Roche)) and incubated on ice for 30 min. After centrifugation (16000 xg, 4°C, 15 min) the supernatant was mixed with SDS-loading buffer and 24 μg of total protein was applied to a 12% SDS-gel. For Western blotting, the monoclonal B-2 antibody was used (sc-9996, Santa Cruz) for detection of GFP. For loading control the monoclonal anti beta-Tubulin (E7) antibody from the Developmental Studies Hybridoma Bank (DSHB) was used. Chemiluminescence images were recorded with a Fuji LAS-3000 mini system and quantification of band intensity was performed using ImageJ.

Sample preparation was performed as described previously.(14 (link)) Briefly, cells were lysed in TENSV buffer (50 mM Tris–HCl (pH 7.5), 2 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, 1 mM Na₃VO₄, 1% NP-40, 0.1% aprotinin, and 2 mM phenylmethylsulfonyl fluoride), and electrophoresed in sodium dodecyl sulfate (SDS)-polyacrylamide gel. The proteins were transferred to a membrane and immunoblotted with an anti-Akt (pan) monoclonal antibody (mAb) (clone C67E7, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Ser473) mAb (clone D9E, Cell Signaling Technology), anti-PDGFRβ polyclonal antibody (P-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phospho-PDGFRβ mAb (clone 42F9, Cell Signaling Technology), and anti-α-tubulin mAb (clone YL1/2, AbD Serotec, Kidlington, UK). The LAS-3000 mini system (Fujifilm, Tokyo, Japan) was used for visualization and quantification of signals.

Tumor tissues were lysed with RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Protein concentrations were measured using BCA protein assay kits (Takara). Proteins were electrophoretically separated on 7.5% SDS‐PAGE gels. Forty micrograms of protein were loaded per lane. Proteins were then transferred to PVDF membranes, and Western blotting carried out to detect HIF‐1α. Anti‐HIF‐1α mouse monoclonal IgG2b (1:500, NB100‐105; Novus Biologicals, Littleton, CO, USA) was used as the primary antibody. Anti‐mouse IgG‐HRP (1:1000; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody. Horseradish peroxidase was detected with the ECL system (Amersham Biosciences, GE Healthcare, Diegem, Belgium) using an imaging densitometer LAS‐3000 mini system (Fujifilm, Kanagawa, Japan).