Ab32042

Protein expressions were determined using Western blot assay [33 (link)]. Total protein was extracted using RIPA cell lysate buffer containing a protease inhibitor cocktail. The protein concentration was then measured using the BCA method. The proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membranes. The membranes were then blocked with 5% BSA at room temperature for 50 min to eliminate nonspecific binding of primary and secondary antibodies. Primary antibody (cleaved-Caspase3 antibody, ab32042, 1:1000, Abcam; GAPDH antibody, ab9485, 1:1000, Abcam; PCNA antibody, ab18197, 1:1000, Abcam; CCND2 antibody, ab207604, 1:1000, Abcam;) was then added to the PVDF membrane and incubated overnight at 4°C. The next day, the membranes were probed with the corresponding HRP-labeled secondary antibody (ab7090, 1:1000, Abcam) by incubation at room temperature for 2 h. ECL chromogenic solution A and B were mixed, added dropwise to the position of the target band on the membrane, and photographed using ImageJ software.

The total protein was extracted using a radioimmunoprecipitation assay, and the bicinchoninic acid (Beyotime, Shanghai, China) method was employed to quantify the concentration. The protein sample was first electrophoresed for 2h; subsequently, the authors transferred the total protein onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). TBST containing 5% skim milk was used to block non-specific antigens for 1h following incubating with primary antibodies (caspase-3 [1:1,000, ab32351; Abcam], c-caspase-3 [1:1,000, ab32042; Abcam], CREB1 [1:1,000, ab32515; Abcam], p-CREB1 [1:1,000, ab32096; Abcam], Bcl-2 [1:1,000, ab182858; Abcam], PSD-95 [1:1,000, ab238135; Abcam], synaptophysin [1:1,000, ab32127; Abcam], synapsin [1:1,000, ab254349; Abcam], GAPDH [1:1,000, ab8245; Abcam], and β-actin [1:5,000, #A5441, Sigma]) at 4°C overnight. Membranes were washed with TBST three times and incubated with the secondary HRP-conjugated goat anti-rabbit IgG (1:5,000 dilution; cat. ab205719; Abcam). Subsequently, membranes were washed with TBST three times. Blots were then visualized using enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK).

After treatment with various doses of tryptamine, PC-3 cells were lysed with RIPA lysis buffer (Beyotime, Shanghai, China) containing 1 mM PMSF, and the total protein concentration was determined by BCA Protein Assay Kit (Solarbio, Beijing, China). Samples were electrophoresed on a 10% sodium dodecylsulfate (SDS) polyacrylamide gel and electrotransferred onto polyvinylidene difluoride (PVDF) membrane (Beyotime, China). After blocking with 5% BSA for 1 h, the membrane was incubated with the primary antibodies rabbit anti-cleaved caspase-3 (1:1000, ab32042, Abcam) and anti-β-actin (1:1000, 4967S, Cell Signaling Technology, Boston, MA, USA) at 4 °C overnight, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody (1:2000, 7074S, Cell Signaling Technology) for 1 h at 37 °C. After washing with TBST, the bands were visualized by the enhanced chemiluminescence kit (Thermo Fisher Scientific, Carlsbad, CA, USA) and Odyssey^® imaging system (LI-COR, Lincoln, NE, USA) according to the manufacturers’ instructions.

Total proteins in HTR-8/SVneo cells after the indicated treatment were isolated with RIPA buffer. The protein concentration was detected by using a bicinchoninic acid (BCA) protein assay kit (Invitrogen, USA). After being subjected to a 12% gel sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then impeded with 5% skim milk for 2 h followed by incubation with primary antibodies against Bcl-2 (ab32124; 1:1000; Abcam, China), Bax (ab32503; 1:1000; Abcam), cleaved-caspase3 (ab32042; 1:500; Abcam), MMP2 (ab92536; 1:1000; Abcam), MMP9 (ab76003; 1:1000; Abcam), E-cadherin (ab40772; 1:10000; Abcam), N-cadherin (ab76011; 1:5000; Abcam), p-p38 (mAb #4511; 1:000; Cell Signaling Technology, USA), p-ERK1/2 (mAb #4370; 1:2000; Cell Signaling Technology), p-JNK (mAb #4668; 1:1000; Cell Signaling Technology), p38 (mAb #8690; 1:1000; Cell Signaling Technology), ERK1/2 (mAb #8544; 1:1000; Cell Signaling Technology), JNK (ab76125; 1:1000; Abcam) and GAPDH (ab8245; 1:500; Abcam) at 4˚C overnight. Thereafter, the membranes were washed with tris-buffered saline and Tween (TBST) for three times, followed by incubation of membranes with HRP-labeled secondary antibody. Finally, the protein bands were captured by improved chemiluminescence (ECL, USA).