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6 protocols using ab80590

1

Western Blot Analysis of PDCD4 and PTEN

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Cells were trypsinized and harvested with 1×PBS, then lysed in cell lysis buffer (Invitrogen) with protease inhibitor cocktail (P-2714, Sigma). Lysate protein concentrations were quantified by the Bradford Assay (Bio-Rad). Lysate aliquots containing 30 μg protein were separated on NuPAGE 4–12% Bis-Tris gels (Invitrogen), transferred to PVDF membranes, blocked with blocking buffer, and incubated with antibodies against PDCD4 protein (ab80590, Abcam), PTEN protein (9552S, Cell Signaling), and β-actin (AM4302, Ambion), followed by incubation with secondary antibodies labeled with horseradish peroxidase (Invitrogen). The resulting protein bands were imaged by luminescence using a SuperSignal West Femto Chemiluminescent Substrate (Thermo Scientific), on a Kodak Image Station 2000R, and analyzed with Molecular Imaging Software version 5.0.2.30 (Carestream).
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2

Immunohistochemical Analysis of PDCD4, Ki-67, and eIF4A1

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IHC was employed on 3 μm formalin-fixed paraffin-embedded sections using anti-PDCD4 (1:200; ab80590; Abcam, Cambridge, MA, USA), anti-Ki-67 (1:100; ab16667; Abcam) and anti-eIF4A1 (1:200; ab31217; Abcam). All sections were subsequently incubated with secondary antibody (Vector Laboratories, Burlingame, CA, USA) and developed in diaminobenzidine (DAB). All sections were then washed in PBS. Appropriate positive and negative controls were included for each relevant stain.
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3

Western Blot Analysis of PDCD4 Protein

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Cells were solubilized in cold radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.) to extract protein. The protein concentration was determined using a bicinchoninic acid Protein Assay kit (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Protein (50 µg) was separated by 10% SDS-PAGE (Pierce; Thermo Fisher Scientific, Inc.), and transferred onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, Inc.). Following incubation with PBS containing 5% non-fat milk at 4˚C overnight, the membrane was incubated with rabbit anti-human PDCD4 primary antibody (1:50; ab80590; Abcam, Cambridge, MA, USA) or rabbit anti-human GAPDH primary antibody (1:100; ab9485; Abcam), respectively, at room temperature for 3 h. Following washing with PBS-Tween-20 for 15 min, the membrane was incubated with the horseradish peroxidase-conjugated mouse anti-rabbit secondary antibody (1:10,000; ab99697; Abcam) at room temperature for 1 h. Chemiluminescent detection was conducted using an Novex™ ECL Chemiluminescent Substrate Reagent kit (Pierce; Thermo Fisher Scientific, Inc). The protein expression was analyzed using Image-Pro Plus software 6.0 (Media Cybernetics, Inc., Rockville, MD, USA), represented as the density ratio vs. GAPDH.
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4

Immunohistochemical Analysis of Tumor Samples

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Tumor samples were harvested and fixed using formalin, followed by paraffin-embedment. Sections were prepared into sections (5-µm thickness) for staining protocol. Sections were first dewaxed in xylene and rehydrated with serial ethanol gradients and washed in deionized water. Subsequently, sections were blocked in PBS with 1% BSA, 1% donkey serum and 0.3% Triton X-100 and 0.01% sodium azide for 1 h, room temperature. After blocking, the slides were exposed to MEK (1:200, Abcam, ab32576), STAT3 (1:200, Abcam, ab68153), PDCD4 (1:200, Abcam, ab80590), and FAP antibody (1:200, Abcam, ab227703) at 4 °C overnight, washed and incubated in biotinylated link universal antiserum for 1 h at room temperature. Primary antibodies were diluted in blocking buffer and incubated in the cold overnight. The next day, sections were washed in PBST 3 times and incubated with secondary antibodies in blocking buffer (1 h, room temperature). Stained sections were then observed under a microscope and micrographed using a mounted digital camera.
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5

Quantifying Proliferation and Apoptosis

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The tissue was fixed by 4% formalin and embedded in paraffin. The endogenous peroxidase activity was blocked, and each slide was subjected to antigen retrieval after peeling and rehydration. The slides were incubated overnight with antibodies against Ki67 (1:500, #ab15580, Abcam) and PDCD4 (1:500, #ab80590, Abcam) at 4 °C. Slides were then incubated with a second antibody coupled with horseradish peroxidase (HRP) at 37 °C for 1 h. The positive immune response rate was determined according to the ratio of positive cells.
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6

Exosomal Protein Expression Analysis

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Protein CD9, CD63 and tumor susceptibility gene 101 (TSG101) were collected from serum exosomes whereas PDCD4, cleaved poly-ADP-ribose-polymerase (cleaved-PARP), cleaved caspase3 (cleaved-casp3) and cleaved caspase9 (cleaved-casp9) were isolated from T24/DDP and 5637/DDP cells. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Mass, USA) and blocked by 5% nonfat milk. Next, the membranes were incubated with primary antibodies against CD9 (ab92726, 1 : 5000), CD63 (ab217345, 1 : 2000), TSG101 (ab125011, 1 : 5000), PDCD4 (ab80590, 1 : 5000), cleaved-PARP (ab32064, 1 : 10 000), cleaved-casp3 (ab2302, 1 : 10 000) and cleaved-casp9 (ab2324, 1 : 5000) or GAPDH (ab181602, 1 : 10 000) (Abcam, Cambridge, MA, USA) and HRP-conjugated secondary antibody (Sangon, Shanghai, China).
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