Ecl western blot kit

Following cell lysate preparation, an equivalent amount of protein lysate (20–25 μg) was electrophoresed on a 4–12% NuPAGE gradient or 7% NuPAGE gels with MOPS buffer or Tris-Acetate Buffer and then electrotransferred to a nylon membrane as described elsewhere [8 (link)]. Protein detection was performed using an ECL western blot kit (Amersham, Arlington Heights, IL, USA). Densitometry was performed using ImageQuant TL software (Amersham) as previously described [34 (link)]. The arbitrary densitometric units for each protein of interest were normalized to the densitometric units for GAPDH.

HCT116 cells were harvested by scraping from the wells and washed twice with cold phosphate buffered saline (PBS). Total cell extracts were prepared using a PRO-PREP^TM protein extraction kit (iNtRON Biotechnology, Seongnam-si, Gyeonggi-do, Korea). Lysates were centrifuged at 16,000 rpm at 4 °C for 30 min to obtain the supernatants. Protein concentration was determined by a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA), loaded on 10% SDS-PAGE, and electrophoretically transferred to a polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies overnight at 4 °C, washed three times, and reincubated with peroxidase-labeled secondary antibody for 2 h. A chemiluminescence (ECL) western blot kit (Amersham, Arlington Heights, IL, USA) was used to detect specific proteins.

Extracts were made from sub-confluent monolayers of cells with RIPA buffer, resolved by 4–20% SDS-PAGE and electroblotted onto a PVDF membrane. The membrane was incubated in PBS, 0.1% Tween-20 and 5% non-fat dry milk powder to block remaining protein binding sites, and then incubated with anti-SSX2-4 (1:3000) (52 (link)) or anti-ZBED1 (Sigma Aldrich, wh0009189M1, 1:2000). The blot was further stained with horseradish peroxidase-conjugated goat anti-mouse IgG (DakoCytomation Denmark A/S, Glostrup, Denmark) and developed with ECL Western Blot kit (Amersham Biosciences, Hilleroed, Denmark, 1:100 000). All antibody incubation and washing steps were carried out in PBS, 0.1% Tween-20.

20 μg protein extract were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk and followed by incubating with the primary antibody (FUT4, AP12067b, Abgent; cleaved caspase-3 ab2302, Abcam; cleaved PARP, ab4830, Abcam; Sp1, ab13370, Abcam; CD44, ab157107, Abcam; GSK-3β, 22,104–1-AP, Proteintech; p-GSK-3β, 22,104–1-AP, Proteintech; β-catenin, 51,067–2-AP, Proteintech; CyclinD1, 60,186–1-Ig, Proteintech; GAPDH, AP7873a, Abgent) on a shaker overnight at 4 °C. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (rabbit IgG, 1/1000 diluted; UK). A GAPDH antibody (1/200 diluted; Santa Cruz Biotech) was used as a control. All bands were detected using ECL Western blot kit (Amersham Biosciences, UK). The bands were measured with LabWorks (TM ver4.6, UVP, BioImaging systems).

Exosomes and cells were lysed using radioimmunoprecipitation assay lysis buffer containing protein inhibitors. An equivalent quantity of proteins (15 μg) was electrophorized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; and then transferred to a polyvinylidene fluoride (Bio-Rad Laboratories, Inc) membrane. After being blocked in 5% (w/v) skimmed milk, the membrane was incubated with primary antibodies overnight at 4 °C. The primary antibodies were: FRMD3 (ab166071, 1:1000), CD-9 (ab92726, 1:2000), CD-63 (ab134045, 1:10000), TSG101 (ab125011, 1:1000), E-cadherin (ab40772, 1:10000), N-cadherin (ab76011, 1:5000), Vimentin (ab92547, 1:1000) and GAPDH (ab9485, 1:2500). Subsequently, the membrane was reacted with horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG H&L (ab6721, 1:2000) for 1h. All antibodies were bought from Abcam Company (UK). All bands were measured by electrochemiluminescence (ECL) western blot kit (Amersham Biosciences, Little Chalfont, UK) and processed by Image Lab 6.0.1 Software (Bio-Rad Laboratories, Inc.).

Based on protein concentration measurements, equal amounts of plasma membrane preparations generated from all cells were resolved by 4–20% SDS-PAGE (Pierce) and transferred onto a PVDF membrane. The membranes were blocked in PBS, 0.1% Tween 20, 5% nonfat dry milk powder for 1 h at room temperature and incubated with primary antibody for 16 h at 4°C followed by washing and incubation with HRP-conjugated secondary antibody for 1 h at room temperature. All antibody incubations and washing steps were carried out in PBS, 0.1% Tween 20. The immunoreactive bands were visualized using an ECL Western blot kit (Amersham Biosciences). The western blots were normalized by Ponceau staining [10 (link)].