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4 protocols using interleukin 6 (il 6)

1

Phosphorylation-mediated signaling analysis

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Primary antibodies: Phospho-STAT3 (Tyr705) (Thermo Fisher, USA; Cat# 44380G), p-NFκB p65 (S536) (Thermo Fisher, USA; Cat# MA515160), STAT3 (Thermo Fisher, USA; Cat# 10253-2-AP), beta Crystallin A3 (Abcam, USA; Cat# ab151722), IL-6 (Biorbyt, USA; Cat# orb6210), IL-1α (Biorbyt, USA; Cat# orb184287) and mNeonGreen (Chromotek, USA; Cat# 3216-100), Secondary antibodies: HRP anti-Rabbit IgG (KPL, USA; Cat# 074-1506), HRP anti-tagged anti-Mouse IgG (KPL, USA; Cat# 5220-0341), HRP anti-tagged Goat IgG (KPL, USA; Cat# 14-13-06). GIPZ Cryba1 shRNA Viral Particle Starter Kit (Dharmacon, USA; Cat# VGH5526-EG12957), AAV2-GFP-U6-shRNA (Vector Biolabs, USA; Cat# 7041). PTP1B (Ad-CMV-mNeonGreen-mPtpn1; Cat# AAV-269791) and Cryba1 (Ad-CMV-RFP-mCryba1; Cat# 2001) overexpression vectors were purchased from Vector Biolabs, USA. ELISA kits: Human crystallin, beta A1 (CRYBA1) (Cat# MBS7252187) and protein tyrosine phosphatase 1B (PTP1B) (Cat# MBS761801) ELISA kits were purchased from Mybiosource, USA. Proteins: CRYBA1 (NM_005208) purified human protein (OriGene Technologies, Cat# TP321965), Recombinant human PTP1B protein (Abcam, Cat# ab51277).
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2

HNSCC-PBMC Coculture Cytokine Analysis

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HNSCC cells (2 × 105) were seeded in 24-well plates overnight. The next day, PBMCs (3 × 106) were cocultured with the HNSCC cells with/without 10 µg/mL CDA for 2 and 6 hours. For transwell cocultures, 2 × 105 HNSCC cells were seeded into the bottom compartment of a 24-well plate overnight. PBMCs (3 × 106) were seeded into the top 0.4-µm ThinCert cell culture insert (Greiner Bio-One, catalog no. 665641) the next day and cultured for 6 hours ± 10 µg/mL CDA. Supernatants were harvested and the levels of IFNβ, IL6, TNFα, and IL1β were assessed with human type-I IFN (Biorbyt, catalog no. orb561974), TNFα (Invitrogen, catalog no. 88–7346), IL6 (Invitrogen, catalog no. 88–7066), and IL1β (Invitrogen, catalog no. 88–7261) ELISA kits following the manufacturer's instructions.
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3

Cytokine Secretion in Prostate Cancer Cells

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DU145 and PC3 cells were seeded onto 24-well plates at a cell count of 2 × 104 cells/well in a regular culture medium and incubated at 37°C for 48 h in the absence and the presence of KPT-330 and KPT- 251 at concentrations as indicated. The conditioned medium was collected and assayed with a sandwich ELISA for TGF-β1 (RayBiotech, Inc., Norcross GA), IL-8 (Biorbyt ltd, Cambridge, UK), IL6 (Biorbyt ltd), VEGF (Biorbyt ltd) and RANKL (Enzo Life Sciences, Inc. Italian distributor Vinci-Biochem Florence) according to the manufacturer’s protocol.
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4

Astrocyte Protein Analysis via Western Blot

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Proteins from astrocytes were obtained using the lysis solution (Biorbyt, Cambridge, UK) . The BCA kit (Biorbyt, Cambridge, UK) was used to quantify the isolated proteins, which were loaded on a gel, separated by 12% SDS-PAGE, and transferred onto the PVDF membrane (Merck, New Jersey, USA) . The membrane was further incubated in 5% skim milk and incubated at 4℃ overnight with primary antibodies against p-JAK2 (1:800, Biorbyt, Cambridge, UK) , p-STAT3 (1:800, Biorbyt, Cambridge, UK) , p-AKT (1:800, Biorbyt, Cambridge, UK) , GP130 (1:800, Biorbyt, Cambridge, UK) , IL-6 (1:800, Biorbyt, Cambridge, UK) , or GAPDH (1:800, Biorbyt, Cambridge, UK) . Then, the membrane was incubated with secondary antibody (1:4000, Biorbyt, Cambridge, UK) for 2 h. Lastly, following exposure, bands were analyzed using the Image J software (Bio-Rad, California, USA) with GAPDH as the loading control.
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