Ultrasensitive ecl chemiluminescence kit

The treated cells were collected and washed twice with PBS buffer. Cells were then lysed by RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) for total protein extraction. The BCA protein assay kit (Beyotime Biotechnology) was applied for determining protein concentration. Protein with equivalent amounts were subsequently separated by SDS/PAGE and then transferred on to a PVDF membrane. After blocking, the membranes were incubated with primary antibodies against CXXC4 (ab105400, 1:500), BCL-2 (ab32124, 1:1000), Bax (ab32503, 1:1000), and β-actin (ab8227, 1:3000) (Abcam, Cambridge, U.K.) overnight at 4°C. After that, the membranes were incubated with secondary antibody (ab205718) (Abcam) and exposed using an Ultrasensitive ECL Chemiluminescence kit (Sangon Biotech) according to the manual.

Western blotting was used to assess the expression of phosphorylated and unphosphorylated PI3K, Akt, and mTOR, as well as P-gp and Survivin proteins in MCF-7/Survivin cells. The cell lines (2 × 10⁵ cells/well) were cultured in 6-well plates and treated with different doxycycline concentrations (0, 2.25, 22.5, 225, and 2,250 nmol/L) for 24 h. Then, cells were lysed with lysis buffer, and the lysate was collected. After SDS-PAGE (12%), the samples were electro-transferred onto a pretreated PVDF membrane. The membrane was blocked using 3% non-fat milk (or 5% bovine serum albumin (BSA)) in Tris-buffer saline with Tween 20 (TBST) for 2 h at room temperature and incubated with antibodies at 4 °C overnight. Further incubation with the secondary antibody was performed for 1 h at room temperature after washing with TBST 3 times. Protein bands were visualized with the ultrasensitive ECL chemiluminescence kit (Sangon Biotech, Shanghai, China). Protein quantification was performed with ImageJ (1.51K).

Algal cells were sedimented and ground to a fine powder with a mortar and pestle in liquid nitrogen. Proteins were extracted from the powder homogenized in an ice-cold extraction buffer consisting of 50 mM Tris–HCl (pH 8.0), 5 mM MgCl₂, 0.1% (v/v) Triton X-100, 10% (v/v) glycerol, 2 mM benzamidine, 2 mM ε-aminocaproic acid, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and 10 mM dithiothreitol. The extract was centrifuged at 12,000×g for 5 min at 4 °C and the supernatant was used for western blot.
Proteins were separated on a 10% (w/v) SDS polyacrylamide gel and blotted onto a polyvinylidene fluoride (PVDF) membrane (GE healthcare, USA). An anti-sFBA antibody, raised in rabbits against synthetic peptides (ETGQGEAEDGHGFEGKC) according to partial sequence of sFBA, was prepared and conjugated with horseradish peroxidase (HRP). The sFBA protein was detected using the HRP-conjugated rabbit anti-sFBA antibody and the HRP-conjugated goat anti-rabbit IgG as the secondary antibody. The antibody-positive bands were detected using Ultrasensitive ECL Chemiluminescence Kit (Sangon Biotech, China). Protein concentration was determined using Bicinchoninic Acid (BCA) Protein Assay Kit (Cwbio Biotechnology, China).