NeuroD1 Syt5 Snap25 Cacna1a Cacnb1 Vamp2 Jnk Forward Primers (five -3 ) GCCTCATCCGAAAGAAGTTGC GTGGTTCCCTCAAGTTTTGC CAGCAATGGTCGGGACATAG0 ATTCTTTCAGAAACGTGTGCCAG CGGGAGTGGTCAAGAATGTG GCCCTCTGCCTGAAATACCTT ACTCCATCAGCAAACCAACC CCACACTGTAAACCAAAGCCC TCACCAGTTCCTCAGTTGTGG AAGGACAGTACCAGGCAGTTC GCCTATTGAAAATCTGCTCTGG TTGTGTCATCAGCGAAAGTGG GAGGAGGAGCGCAAGATCGG CCCTAACTGATTGCACCAGC CACCTGACCCCAGATCCTTT GGCGTTTGCTGAATGACAAC CTAGCCCTGCCAAGATCGG CTTTACCCCAGCAACCACCC TGGTGGACATCATGAGGGTG TCCAGTTCTCGTACCCGCTA Reverse Primers (5 -3 ) TGGCCTCAGAGAAACCTAGGA GTGCTGCAGATAATGAGGGCA AGACTGCCCATTCTCGACAAG ATCCCCATTTTCATCCTTCC CATGAGCTTGAGAGTTTCCTGC CAGGGTCCTTCTTTGGCAGAT CCTCGATTTCTGGGCAGTTCT GGAAGGCCTCGAATGACATCA GCACGGCAGAGTTTTCAGTTT CCAAGTAGGGCTGTGTTTGC ATTGCTCTGAATGACTCTGG CACAGGACTAGAACGTCTGCT AGCAAAAGTTTCGTGCTGTCAA TGCAGGGTAGTGCATGGTAA GAGTGGTACTGGAAGTCGGA CAGAGCCTGACACCCTAAGA ACGATAAGGCTGTTCTCGG GTCCACACACGAGTCTCCTG GCTTGGCTGCACTTGTTTCA AGCATGGCGTGACACAGTA4.5. Insulin Secretion Assay GSIS measurements had been performed 48 h post-transfection, as previously described [58]. 1st, INS-1 (832/13) -cells had been incubated in pre-warmed secretion assay buffer (SAB) with 2.8 mM glucose for 2 h. The cells had been then stimulated with SAB containing either 2.eight mM glucose, 16.7 mM glucose, 2.8 mM glucose plus ten mM -KIC, or 35 mM KCl for 1 h. Subsequent, the amount of secreted insulin was determined applying the rat insulin ELISA kit (Mercodia, Uppsala, Sweden) and normalized for the total amount of protein. 4.six. Western Blot Analysis To detect activated inflammasome proteins, INS-1 cells were stimulated with 1 LPS/200 PA SA for four h [24,59]. Total protein extraction was performed working with ice-cold NP-40 (1.Fmoc-N-Me-Phe-OH Purity 0 NP-40, 150 mM NaCl, 50 mM Tris-Cl, pH eight.0) lysis buffer containing a protease inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA).Formula of 4-Acryloylmorpholine A Western blot evaluation was performed as previously described [58] together with the following antibodies: MAPK8IP1 (antirabbit; 1:1000, #Ab24449, Abcam, Cambridge, UK), NLRP3 (anti-rabbit; 1:1000, #A12694, Abclonal, Woburn, MA, USA), CASPASE-1 (anti-rabbit; 1:1000, #A0964, Abclonal, Woburn, MA, USA), IL-1 (anti-rabbit; 1:1000, #A162888, Abclonal, USA), GSDMD (anti-rabbit; 1:1000, #A10164, Abclonal, USA), JNK (anti-rabbit; 1:1000, #A48567, Abclonal, USA), pJNK (anti-rabbit; 1:1000, #AP0631, Abclonal, USA), -actin (anti-mouse, 1:1000, #A5441, SigmaAldrich, Darmstadt, Germany), and secondary anti-mouse (#7076S) or anti-rabbit (#7074S, from Cell Signaling Technologies, Danvers, MA, USA).PMID:33749465 Chemiluminescence was detected applying the Clarity ECL substrate kit (Bio-Rad, Hercules, CA, USA). -actin was utilized as an endogenous handle. 4.7. Apoptosis Assay The transfected and non-transfected cells have been cultured in RPMI medium within the presence of vehicle (control) or 1 LPS followed by 200 PA SA, as described earlier. Following 24 h incubation, the cells were re-suspended in 500 of Annexin-V (1X) BindingInt. J. Mol. Sci. 2023, 24,15 ofBuffer (BD Biosciences, San Jose, CA, USA) after which stained with two of Annexin V-FITC and two of Propidium Iodide (PI) (15 min) in the dark. The cells have been analyzed working with a BD FACS Aria III flow cytometer (Becton Dickinson, Biosciences, Franklin Lakes, NJ, USA). 4.eight. Cell Viability Assay An MTT colorimetric assay (Sigma-Aldrich, Saint Louis, MO, USA) was applied to assess cell viability. In short, transfected and non-transfected INS-1 (832/13) cells, seeded in 96-well plates (20 ?104 /well), have been cultured in RPMI medium inside the.
