β tubulin

Brains were quickly removed after decapitation. Striatum and midbrain samples were obtained at 0 °C and subsequently homogenized using Disposable Pellet Mixers with Pestle Motor (VWR). Tissue homogenates were prepared in RIPA buffer; pH 7.5, containing 10% Triton 500 μl, 5 μl aprotinin, PMSF 50 μl, Na₃VO₄ 100 μl and NaF 20 μl in 4.32 ml PBS. Extracts were clarified by centrifugation at 4 °C (13,200g for 20 min). Supernatants were collected and eluted with RIPA buffer, and the proteins were resolved by SDS-PAGE [45 (link)]. The primary antibodies used were: β-Tubulin (#05-661, Millipore), β-Actin (Abcam, ab6276, AC15), HA-Tag (C29F4) (#3724, Cell Signalling), Tyrosine Hydroxylase (MAB318, Sigma Aldrich), α-synuclein (610787, BD Transduction Laboratories™), DAT (MAB369, Merckmillipore), LC3 (5F10, Nanotools Antibodies), Tom20 (D8T4N) (#42406, Cell Signalling), Mitofusin-2 (D2D10) (#9482, Cell Signalling), DRP1 (D6C7) (#8570, Cell Signalling), Phospho-Tau Ser202/Thr205 (AT8) (#MN1020, ThermoFisher), and Dopamine D2 Receptor (AB5084P, Merckmillipore). The rabbit anti-mouse (GE healthcare UK, NA934V), or mouse anti-rabbit (GE health UK, NA931V) were used to react with the corresponding primary antibodies. Immunoreactive bands were visualized by enhanced chemiluminescence (GE Healthcare). Densitometry analysis on the bands was calculated using ImageJ software.

SDS-PAGE and Western blotting were performed using whole cell lysates, or cytoplasmic or nuclear fractions prepared from 100,000 cells/well. The following antibodies were used to probe the membrane: Ago2 (11A9, [74 (link)]), β-Tubulin (Millipore, CP06-100UG), Sm antigen (Dr. Joan Steitz’s lab, Yale University), Lamin A/C (Active Motif, 39288), Calnexin (ENZO Lifesciences, ADI-SPA-865-D).

Antibody specific to phospho-CEP55 (S436) was raised against the synthetic phosphopeptide CKRIFKDLGTPFAL(pS)KSSM. Phospho-specific antibody was obtained though two-step affinity-purification methods. Commercially available antibodies for WB were as follows: ASPP1 (ab137537; Abcam), ASPP2 (611354; BD Biosciences), iASPP (18590-1-AP; Proteintech), CEP55 (23891-1-AP; Proteintech), CEP55 (7825-1; epitomics), α-Tubulin (1878-s; epitomics), β-Tubulin (05–661; Millipore), ALIX (sc-67338; Santa Cruz), TSG101 (4A10; Genetex), CHMP2B (12527-1-AP; Proteintech), CHMP4B(13683-1-AP; Proteintech), MKLP-1 (sc-867; Santa Cruz), MgcRacGAP (H00029127; Novus), PP1α (1950–1; epitomics), PP1β (2029–1; epitomics), PP1γ (6646–1; epitomics), Myc (9E10; Sigma), FLAG (M2; Sigma), HA (MM5-101R; Millipore), and Actin (AC-74; Sigma).

SNU-16 parental and resistant cells were seeded in six-well plates at a density of 0.5 × 10⁶ cells per milliliter in an inhibitor-free medium. After 24 h, cells were lysed and the level of proteins was examined by Western blotting according to the protocols provided by the antibody suppliers. The antibodies against MET, EGFR, HER2, extracellular-signal-regulated kinase (ERK), phosphorylated AKT, phosphorylated EGFR, phosphorylated ERK (pERK), phosphorylated FGFR, phosphorylated FGFR substrate, and phosphorylated signal transducer and activator of transcription (STAT3) were purchased from Cell Signaling Technology, β-tubulin and AKT were from Millipore, E-cadherin, STAT3, and β-catenin were from BD Biosciences, FGFR2 was from Abnova, and vimentin was from Calbiochem. All experiments were performed at least twice.

100 μg of total protein was extracted from 2 dpf embryos and electrophoresed through a 12.5 % polyacrylamide SDS gel. Protein was transferred to a nitrocellulose membrane and probed with either; anti-α-actin (Sigma clone A2066, 1:400) or β-tubulin (1:5000, Millipore). Secondary HRP antibodies were all used at 1:5000 (Chemicon).