Cells were treated with test compounds and, after difierent times, collected, centrifuged, and washed with ice cold phosphate buffered saline. The pellet was then resuspended in lysis buffer as described.25 (link) The protein concentration in the supernatant was determined using the BCA protein assay (Pierce, Milano, Italy). Equal amounts of protein (10–20 μg) were resolved using SDS–PAGE gel electrophoresis (Criterion precast Tris-HCl gel, Biorad Laboratories, Milano, Italy) and transferred to PVDF Hybond-p membranes (GE Healthcare, Milano, Italy). Membranes were blocked with 2% ECL-blocking solution (GE Healthcare, Milano, Italy) for 2 h, with rotation at room temperature. Membranes were then incubated with primary antibodies against Bcl-2, p53, PARP, procaspase-9, procaspase-8, procaspase-2, Mcl-1, Bcl-XL (Cell Signaling, Milano, Italy), β-actin (Sigma-Aldrich, Milano, Italy), and caspase-3 (Novus Biologicals, Milano, Italy) overnight. Membranes were next incubated with peroxidase-conjugated secondary antibodies (Invitrogen, Milano, Italy) for 60 min. All membranes were visualized using ECL Select (GE Healthcare, Milano, Italy) and exposed to Hyperfilm MP (GE Healthcare, Milano, Italy). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.
Hybond p pvdf membrane
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Italy, Japan, Brazil, Sweden
Hybond-P PVDF membrane is a polyvinylidene difluoride (PVDF) membrane used in various laboratory applications. It serves as a support matrix for protein transfer and immobilization in techniques such as Western blotting. The membrane provides a high-binding capacity for proteins and is compatible with a range of staining and detection methods.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (101 mentions)
Las 4000, by Cytiva (17 mentions)
Imagequant las 4000 mini digital imaging system, by GE Healthcare (9 mentions)
β actin, by Merck Group (6 mentions)
Ecl select, by GE Healthcare (6 mentions)
Mes buffer, by Thermo Fisher Scientific (6 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (5 mentions)
Semi dry apparatus, by Hoefer (5 mentions)
Hyperfilm mp, by GE Healthcare (4 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (4 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (4 mentions)
Nupage 4 12 bis tris gradient gel, by Thermo Fisher Scientific (4 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (3 mentions)
Las 4000 mini luminescent image analyzer, by GE Healthcare (3 mentions)
Ecltm western blotting detection reagent, by GE Healthcare (3 mentions)
Hrp linked whole antibody of sheep anti mouse igg, by GE Healthcare (3 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
104 protocols using hybond p pvdf membrane
1
Western Blot Analysis of Cell Lysates
2
Quantitative Western Blot Analysis
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Cells were treated with CP and, after different times, were collected, centrifuged and washed with ice cold phosphate-buffered saline (PBS). The pellet was then resuspended in lysis buffer as described [47 ]. The protein concentration in the supernatant was determined using the BCA protein assay (Pierce, Italy). Equal amounts of protein (10 μg) were resolved using SDS-PAGE gel electrophoresis (Criterion precast Tris-HCl gel, BioRad, Italy) and transferred to PVDF Hybond-p membranes (GE Healthcare, Italy). Membranes were blocked with 2% ECL-Blocking Solution (GE Healthcare, Italy) for 2 hours at room temperature. Membranes were then incubated with primary antibodies against Bcl-2, PARP, procaspase-9, cleaved caspase-7, GRP78, (Cell Signaling, Italy), cleaved caspase-12 (Abcam, UK), β-actin (Sigma Aldrich, Italy), and cleaved caspase-3 (Novus Biologicals, Italy) overnight. Membranes were then incubated with peroxidase-conjugated secondary antibodies (Invitrogen, Italy) for 60 min. All membranes were visualized using ECL Select (GE Healthcare, Italy) and exposed to Hyperfilm MP (GE Healthcare, Italy). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody.
3
Anthocyanin Effects on Protein Expression
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The effect of the ANC from purple corn on selected proteins expression was assessed by western blot. iNS-1E and HepG2 cells were cultured in dual-layered system as described in the cell culture and dual-layered cell culture system section. The cells were seeded in the basolateral side of the system in 6-well plates. On the day of the experiment, Caco-2 cells were apically treated with 100 μM of pure ANC (C3G, P3G, Pr3G, D3G, C3G-P, and CF-P) or 0.5 mg/mL of the anthocyanin-rich extract (PCW) for 24h. Subsequently, the cells were lysed with RIPA buffer; lysates were separated through SDS-PAGE and transferred to PVDF Hybond-P membranes; primary and secondary (GE Healthcare, Buckinghamshire, UK) antibodies were used following manufacturer’s recommended dilutions for western blot. Protein expression was detected using 1:1 chemiluminescent reagents of ECL Prime Western Blotting kit (GE Healthcare, Buckinghamshire, UK) and visualized using a Gel Logic 4000 Pro Imaging System. After ECL detection, the membranes were washed, stripped with Restore PLUS Western Blot stripping buffer (Thermo Scientific, Lafayette, CO), re-blocked, and re-probed with GAPDH antibody. The intensity of each band was normalized to GAPDH (sc-47724), and the results were expressed as expression level relative to a control.
4
Merecidin-Induced Apoptosis in A549 Lung Cancer Cells
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The lung cancer A549 cells were cultured for 24 h until 80-90% con uency was achieved. The cells were treated with 9 mmol/L merecidin 6 h and observed under a light microscope. The cells were shrunk, their nuclear membranes thickened, or shattered and dissolved. An equivalent of 10-20 mg protein was resolved using SDS-PAGE and transferred to PVDF Hybond-p membranes (GE Healthcare, Milano, Italy). Then, the membranes were probed with primary antibodies against ULK1 (1:1,000 dilution in TBST and 5% skim milk; Abcam, UK), MAPK1 (1:2,000; Abcam), ATG13 (1:1,000; Abcam),and b-tubulin (1:2,000; Abcam) at 4°C overnight. Subsequently, the membranes were incubated with peroxidase-conjugated A nipure goat antirabbit LgG (H + L) (1:1000) secondary antibodies at room temperature for 1 h. The immunoreactive bands were visualized using ECL Advance (GE Healthcare)., and the intensity quanti ed using Image Lab v3.0 software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
5
Merecidin-Induced Apoptosis in A549 Lung Cancer Cells
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The lung cancer A549 cells were cultured for 24 h until 80-90% con uency was achieved. The cells were treated with 9 mmol/L merecidin 6 h and observed under a light microscope. The cells were shrunk, their nuclear membranes thickened, or shattered and dissolved. An equivalent of 10-20 mg protein was resolved using SDS-PAGE and transferred to PVDF Hybond-p membranes (GE Healthcare, Milano, Italy). Then, the membranes were probed with primary antibodies against ULK1 (1:1,000 dilution in TBST and 5% skim milk; Abcam, UK), MAPK1 (1:2,000; Abcam), ATG13 (1:1,000; Abcam),and b-tubulin (1:2,000; Abcam) at 4°C overnight. Subsequently, the membranes were incubated with peroxidase-conjugated A nipure goat antirabbit LgG (H + L) (1:1000) secondary antibodies at room temperature for 1 h. The immunoreactive bands were visualized using ECL Advance (GE Healthcare)., and the intensity quanti ed using Image Lab v3.0 software (Bio-Rad Laboratories Inc., Hercules, CA, USA).
6
Signaling Pathways in CASKI Cells
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In experiments analyzing the signaling pathways, cells were starved for 16h and treated with cetuximab (100 μg/ml) for 90 min, when indicated. Afterward, cells were stimulated with PAF (100 nM) for 5 to 20 min. In experiments to evaluate COX-2 protein expression, the same protocol was used. However, treatment with PAF lasted 3 h. In experiments analyzing PAFR downregulation, CASKI cells were simultaneously starved and treated with cetuximab (100 µg/ml) for 24 h and 48 h. Following treatment completion, cells were lysed, and proteins were quantified using the Lowry method (DC protein assay, Bio-Rad, CA, USA). Protein lysates (20–30 μg) from each condition were subjected to 8%–10% SDS–PAGE and transferred onto a PVDF Hybond-P membrane (GE Healthcare, Brazil). Membranes were blocked and incubated overnight with p-ERK1/2 (Cell Signaling Technology, MA, USA), ERK1/2 (Cell Signaling Technology), COX-2 (Cell Signaling Technology, MA, USA), PAFR (Abcam, MA, USA) and β-actin (Cell Signaling Technology) primary antibodies. Subsequently, membranes were incubated with HRP-conjugated secondary antibodies (DakoCytomation, Denmark) for 1 h at room temperature, and immunoblots were detected using the ECL reagent (GE Healthcare, Brazil).
7
Western Blot Analysis of Protein Targets
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Total protein extracts were isolated in lysis buffer as previously described [22 (link)]. Equal amounts of proteins (10–20 μg) were resolved using SDS-PAGE gels and transferred to PVDF Hybond-p membrane (GE Healthcare). Membranes were blocked with I-block (Life Technologies) for at least 2 hours, under rotation at RT. Membranes were then incubated overnight at 4°C under constant shaking with the following primary antibodies: HIF-1α (mouse, 1 : 250, BD Pharmingen), mTOR total, mTOR (S2448), P70S6K total, P70S6K (T389), AKT total, AKT (T308), AKT (S473), BAX, PARP, (all rabbit, 1 : 1000, Cell Signaling Technologies), and β-actin (mouse, 1 : 10000, Sigma Aldrich) as loading control. Membranes were next incubated with peroxidase-labeled goat anti-rabbit IgG or goat anti-murine IgG (both 1 : 50.000 in I-block from Sigma Aldrich) for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and exposed to Hyperfilm MP (GE Healthcare).
8
Western Blot Analysis of Cell Signaling Proteins
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REH, SEM, MHH-CALL2, RS4;11 and NALM-6, after experimental conditions, were collected, centrifuged, and washed two times with ice cold phosphate-buffered saline (PBS). For western blot analysis cells were lysed as described previously (23 (link)). Proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) (using 7 or 10% acrylamide gels), transferred to PVDF Hybond-P membrane (GE Healthcare) and immunoblotted with primary antibodies against, FOXM1 (Santa Cruz, C-20), β-tubulin (Santa Cruz), Aurora B (Cell Signaling), Cyclin B1 (Santa Cruz). The membranes were washed four times with Tris-buffered saline and Tween-20 (TBS-T) for 15 min prior to incubation with the respective peroxidise (HRP)-conjugated secondary antibody (Dako, Ely, UK), at 1:2,000 dilution for 30 min at room temperature and again washed four times with TBST-T for 20 min. Protein were visualised using enhanced chemiluminescence (ECL) detection system (Perkin-Elmer, Seer Green, UK) with Amsterdam Hyperfilm ECL (GE Helthcare, Little Chalfont, UK) and signal was detected using the SRX-101A X-ray developer (Konica Minolta, Tokyo, Japan).
9
MAPK Signaling Activation by TR47 and TR41
10
Western Blot Analysis of Apoptosis Markers
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