Bsa standard

The resuspended Euglena cells from the culturing (1 ml) were diluted with 5x binding/wash buffer (0.25 ml) containing phenylmethylsulfonyl fluoride (2 mM) and lysed by sonication (3 × 10 s, 25% amplitude, 30 s off between each pulse) and centrifuged (5 min, 1000 × g). Not all cells were lysed. Total lysate containing the equivalent of 1.1 mg of protein (Easy Bradford BioRad, BSA standards) was then used for glycoprotein purification using both ConA and wheat germ agglutinin (WGA) Glycoprotein Isolation Kits (Thermo Scientific) according to the manufacturer’s instructions. Protein quality was assessed using silver-stained SDS-PAGE (Bolt 4%–12% Bis-TRIS plus, Invitrogen) using SeeBlue Plus2 Prestained Protein Standard (Thermo Fisher Scientific) as the standard.

The cells were lysed in buffer containing 20 mM Tris, pH 7.4, 2 mM EGTA, 2 mM Na₂VO₃, 2 mM Na₄P₂O₇, 2% SDS, 2% Triton X-100, 1 μM leupeptin, 1 μM aprotinin, and 1 mM PMSF. We used a protein assay kit by BSA standards to determine the protein concentration according to the manufacturer’s instructions (Bio-Rad Laboratories, Hercules, CA, USA). The amounts of cell lysate were distributed equally via SDS polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech Inc., Oakville, ON, USA). After blocking with Tris-buffered saline (TBS) containing 5% nonfat dry milk for 1 h, we incubated the membranes at 4 °C overnight, with anti-GnRH-I receptor (Neomarker, Fremont, CA, USA), polyclonal total EGFR antibody (Cell Signaling Technology, Danvers, MA, USA), and anti-N-cadherin (Millipore, Darmstadt, Germany) or anti-Twist (Thermal, MA, USA) antibody, followed by incubating them with HRP-conjugated secondary antibody. We read the immunoreactive bands with an enhanced chemiluminescence (ECL) kit. The membrane was stripped by stripping buffer (62.5 mM Tris, 10 mM DTT, and 2% SDS, pH 6.7) at 50 °C for 30 min and re-probed with anti-β-actin antibody (Santa Cruz, Dallas, TX, USA) as a loading control.

The cells were lysed in buffer containing 20 mM Tris, pH 7.4; 2 mM EGTA; 2 mM Na₂VO₃; 2 mM Na₄P₂O₇; 2% Triton X-100; 2% SDS; 1 μM aprotinin; 1 μM leupeptin and 1 mM PMSF. The protein concentration was determined with a protein assay kit using BSA standards according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA). Equal amounts of cell lysate were separated by SDS polyacrylamide gel electrophoresis (PAGE) and transferred to a nitrocellulose membrane (Hybond-C, Amersham Pharmacia Biotech Inc., Oakville, ON). Following blocking with Tris-buffered saline (TBS) containing 5% non-fat dry milk for 1 h, the membranes were incubated overnight at 4°C with anti-GHRH-R (Abcam, Cambridge, MA), anti-Twist (Thermal), or anti-N-cadherin (Millipore) antibody, followed by incubation with a HRP-conjugated secondary antibody. The immunoreactive bands were detected with an enhanced chemiluminescence (ECL) kit. The membrane was then stripped with stripping buffer (62.5 mM Tris, 10 mM DTT, and 2% SDS, pH 6.7) at 50°C for 30 min and re-probed with an anti-GAPDH antibody (Santa Cruz) as a loading control.

For isolation of total lysates, cells were pelleted after 1× PBS wash and lysed in RIPA lysis buffer (Thermo #89900) supplied with protease inhibitor cocktail (Sigma #11836170001). Lysates were collected in new pre‐chilled tubes and protein concentration was measured using Bradford reagent (Bio‐Rad #500‐025) and BSA standards (Bio‐Rad #500‐027). Equal amount of protein was mixed with 4× gel loading dye (Thermo #NP007) and analyzed by pre‐casted gel (Thermo #NP0036) and Western blotting. Final western blots were detected on a GE Healthcare Life Sciences, Amersham Imager 600, followed by analyzing band intensities with ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA).
Antibodies used for western blotting were as follows: anti‐LRP6 (#S2560) primary antibody was purchased from Cell Signaling Technology, antibody against anti‐GAPDH (#GTX627408) was from GeneTex, anti‐Vinculin (#sc‐736914) primary antibody was from Santa Cruz, antibody against β‐catenin (#610153) was from BD Biosciences, anti‐MCL‐1 (#ab32087), anti‐BCL‐2 (#ab692) primary antibodies were from Abcam.

Whole cell extracts from ES Cells and SHTdh striatal cells were isolated using RIPA buffer that contained Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Protein concentrations were measured using Bio-Rad Protein Assay against BSA standards (Bio-Rad, Hercules, CA). Protein from each sample was resolved by SDS-PAGE. Primary antibodies used: Sp1 (Custom-made, 1:1000) and beta-tubulin (ab6046, Abcam). HRP conjugated secondary antibodies (Pierce, ThermoFisher Scientific, Waltham, MA) were used at a dilution of 1:5000. Western Lightning Plus–ECL (PerkinElmer, Waltham, MA) was used for chemiluminescent detection.