Aldh1a1

Small pieces of human lung samples were homogenized and lysed with cell lysis buffer and followed by measurement of protein concentrations. Equal amounts of protein were fractionated by electrophoresis and then transferred to PVDF membranes. The membranes were then incubated with antibodies against NRF2 (Santa Cruz), ALDH1A1 (Abcam), and ALDH3A1 (Santa Cruz) followed by incubation with secondary antibodies. For the detection of specific protein bands, the membranes were incubated in LumiGLO reagent and peroxide (Cell Signaling Technologies, Danvers, MA, USA), and then exposed to X-ray films.

Antibodies to EpCAM (1:100 dilution), CD133 (1:100 dilution), CD44V6 (1: 50 dilution), and ALDH1A1 (1:50 dilution) were purchased from Abcam (Abcam, Cambridge, United Kingdom). Antibody to CD44v8-10 (1:50 dilution) was purchased from Cosmo Bio (Cosmo Bio, Tokyo, Japan).

The anti-TM4SF4 antibody for the Western blot analysis was purchased from Sigma-Aldrich. Antibodies against Sox2, Phospho-AKT (Ser473), AKT, Phospho-NF-κB p65 (Ser536), NF-κB, Phospho-IGF1Rβ (Tyr1131), IGF1Rβ, integrin αV, CD44, and β-actin (Cell Signaling Technology, Danvers, MA, USA); Twist, c-Myc, Cyclin D1, β-catenin (Santa Cruz, Dallas, TX, USA), E-cadherin, and N-cadherin (BD Biosciences, San Jose, CA, USA); Vimentin (Thermo Fisher Scientific, Fremont, CA, USA); and Snail, Notch2, HSPA1L, ALDH1A1, ALDH1A3 (Abcam, Cambridge, UK), and Oct4 (Millipore, Billerica, MA, USA) were used. Protein concentrations were determined using a Lowry kit (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were separated on 8% or 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to a nitrocellulose membrane (Hybond; Amersham Pharmacia). The blots were blocked for 1 h at room temperature with blocking buffer (10% nonfat milk in PBS containing 0.1% Tween 20). The membrane was incubated overnight in a cold chamber with specific antibodies. After being washed with TBS, blots were developed with a peroxidase conjugated secondary antibody, and proteins were visualized via enhanced chemiluminescence procedures (Amersham) following the manufacturer’s protocol.

Cells were grown to ∼70% confluence and reagents were added as indicated. Western blot analysis was performed as described previously (36 (link),37 (link)) with the following antibodies: phospho-SRC (Y416), phospho-STAT3 (T705), and SRC (Cell Signaling Technology, Inc., Boston, MA, USA), ALDH1A1 (Abcam, Cambridge, UK), Poly-ADP-ribose-polymerase (PARP; BD Biosciences, Franklin, NJ, USA), α-tubulin and β-actin (Sigma, St. Louis, MO, USA).