Anti h ras antibody

Proteins (20–30 µg) were electrophoresed on acrylamide gel at 150 V for 1 h and transferred to PVDF membrane at 100 V for 1 h. Rabbit polyclonal anti-H-Ras antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, USA), goat polyclonal anti-TFEB antibody was purchased from Abcam (Cambridge, UK). As internal control, membrane was probed with mouse monoclonal anti β-actin (Sigma-Aldrich, St Louis, USA) and nuclear extracts were normalized with rabbit polyclonal anti-H3 antibody (Millipore, Billerica, USA). Sheep anti-goat (Sigma), donkey anti-rabbit and sheep anti-mouse HRP-linked secondary antibodies (GE Biosciences, Piscataway, USA) were used according to manufacturer’s instructions. Immunoblots were detected by chemiluminescence using ECL (GE Biosciences).

α-SMA and H-Ras proteins expressions in mouse skin were analyzed. Skin samples were mixed in RIPA lysis buffer. Proteins (30 μg per well) were subjected to 10% polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes, blocked with 5% nonfat dry milk in TBS-T, then incubated overnight at 4 °C with an anti-mouse α-SMA (Abcam, Cambridge, UK. ref ab5694, dilution 1:500) or an anti-H-Ras antibody (Santa Cruz Biotechnology, Dallas, TX, USA. ref sc-53959, dilution 1:500). The membranes were then washed and incubated for 1 h with an HRP-conjugated antibody (Invitrogen, Carlsbad, CA, USA) (goat anti-rabbit ref A27036 (dilution 1:5000), goat anti-mouse ref A28177 (dilution 1:10,000)). Tubulin and B actin were used to standardize the OD values obtained for the different samples.

Cell line authenticity was confirmed using the Short Tandem Repeat (STR) Identifier kit (Applied Biosystems). HaCaT cells were cultured in W489 media consisting of 80% MCDB153 and 20% L15 medium supplemented with 1% FBS and maintained at 37°C in a humidified incubator with 5% CO₂. All primary and secondary antibodies were purchased from Cell Signaling Technology (Boston, MA) except anti-HRAS antibody (Santa Cruz Biotechnology, Inc.). Cetuximab (Bristol-Myers Squibb, Princeton, NJ) was purchased from Johns Hopkins Pharmacy. Gefitinib was purchased from Tocris Bioscience (Ellisville, MO). Afatinib (BIBW2992) was purchased from Sellekchem. HaCaT, a spontaneously immortalized keratinocyte cell line was purchased from Cell Lines Service Germany. HaCaT cells were treated with Cetuximab (100nM) and Gefitinib (100nM), as described previously [10 (link), 62 (link)], and afatinib (10nM) based on the dose-response curves for HaCaT-Mock cells. HNSCC cells are grown and treated with Cetuximab (100 nM), as previously described [10 (link), 62 (link)].