Tris 2 carboxyethyl phosphine hydrochloride solution

After UVA irradiation, the CE layer was isolated by removing corneal epithelium and stroma under microscope. The harvested CE layer or cells were, respectively, homogenized in prechilled tissue protein extraction reagent (T-PER, Thermo Fisher Scientific) or mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific) both with protease/phosphatase inhibitor cocktail (cOmplete Mini, IN, USA). Protein concentration was measured by Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific). The samples were heated in NuPAGE Sample Buffer (Thermo Fisher Scientific) containing 5% Tris (2-carboxyethyl) phosphine hydrochloride solution (Sigma–Aldrich) at 95 °C for 5 min. Electrophoresis performed with NuPAGE Bis-Tris Gels (Thermo Fisher Scientific) at 120 V. Next, the transferred polyvinylidene difluoride membranes (Sigma–Aldrich) were blocked and incubated with primary antibodies at 4 °C overnight followed by labeling with HRP-conjugated secondary antibodies at room temperature for 1 h. The incubated membranes were developed with HRP substrate reagent (Genetex, CA, USA) and recorded with an ECL imaging system ChemiDoc MP (Bio-Rad Laboratories, CA, USA).

The GFP-CaMKII beta construct was obtained from Addgene (Addgene Cat #21227). The His-tagged, wild-type (WT) CRABP1 DNA construct for bacterial expression and subsequent protein purification was generated as described in Wook Park et al. (2019) (link). The Flag-tagged, WT CRABP1 DNA constructs for mammalian expression used in HEK293T CRABP1-CaMKII cell assays were described in Wook Park et al. (2019) (link). Constructs of alanine point mutants of His-tagged CRABP1 and Flag-tagged CRABP1 were generated using the Q5^® Site-Directed Mutagenesis Kit (New England BioLabs Inc., Cat #E0554S) according to the manufacturer’s instructions. To validate successful site-directed mutagenesis, the relevant regions of the CRABP1 insert from each mutant construct were validated by Sanger sequencing, which was performed by the University of Minnesota Genomics Center Facility (Minneapolis, MN).
Chemical reagents utilized in this study are as follows: Tris-d11 solution (Sigma Cat # 486248), sodium acetate-d3 (Sigma Cat # 176079), dithiothreitol (DTT) (Gold Biotechnology Cat# DTT10), Tris (2-carboxyethyl)phosphine hydrochloride solution (Sigma Cat # 646547), dimethyl sulfoxide (DMSO) (Sigma Cat #D8418), and ionomycin salt (Sigma Cat #I0634). Ionomycin for molecular and cell studies was prepared by dissolving it in DMSO.

P-PER™ Plant Protein Extraction Reagent, a TMT10plex Isobaric Label Reagent Set, and a Pierce™ BCA Protein Assay Kit were purchased from Thermo Fisher Science (Waltham, MA, USA). Urea, triethylammonium bicarbonate buffer (1.0 M, pH 8.5 ± 0.1), tris (2-carboxyethyl) phosphine hydrochloride solution (0.5 M, pH 7.0), iodoacetamide (IAA), formic acid (FA), acetonitrile (ACN), and methanol were purchased from Sigma (St. Louis, MO, USA). Trypsin from bovine pancreas was purchased from Promega (Madison, WI, USA). Ultrapure water was prepared using a Millipore purification system (Billerica, MA, USA). We also used a Dionex Ultimate 3000 Nano LC system coupled with an Obitrap Q Exactive™ HF mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) with an ESI nanospray source.

RPMI 1640, l-glutamine, streptomycin, penicillin, glutaraldehyde, lipopolysaccharide (LPS), Phorbol 12-myristate 13-acetate (PMA), Tris(2-Carboxyethyl)phosphine hydrochloride solution (TCEP), iodoacetamide (IAA), Bradford reagent, BSA, acetonitrile (ACN) trifluoroacetic acid (≥99.5 %, TFA), and chitosan (with >75 % deacetylation, #417963), were purchased from Sigma-Aldrich (Sigma, St. Louis, MI, USA). With the following materials purchased elsewhere: HEPES and FCS (PAA, Pasching, Austria), SYBR Green Supermix (Bio-Rad), RevertAid H Minus M-MulV reverse transcriptase (MBI Fermentas, St. Leon-Roth, Germany), TRIzol reagent (Invitrogen), CCL2, TNF-α and IL-6 ELISA kits (PeproTech), IL-1-β ELISA (R&D Systems), caspase-1 inhibitor Ac-YVAD-CMK (Calbiochem), epon (Agar Scientific), osmium tetroxide (Electron Microscopy Sciences, Hatfield, UK), triethylammonium bicarbonate buffer (TEAB) (Fluka, Buchs, Switzerland). While trypsin, the LDH detection kit CytoTox 96^® Non-Radioactive Cytotoxicity Assay, and the CellTiterBlue^® (CTB) Cell Viability Assay were purchased from Promega (Madison, WI, USA).

After UVA irradiation, the cells were respectively homogenized in pre-chilled mammalian protein extraction reagent (M-PER, Thermo Fisher Scientific) with protease/phosphatase inhibitor cocktail (cOmplete Mini, IN, USA). Samples concentrations were measured by Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific). The samples were heated in NuPAGE Sample Buffer (Thermo Fisher Scientific) containing 5% Tris(2-carboxyethyl) phosphine hydrochloride solution (Sigma-Aldrich) at 95 °C for 5 min and electrophoresed using NuPAGE Bis-Tris Gels (Thermo Fisher Scientific) at 120 V. Next, the transferred polyvinylidene difluoride membranes (Sigma-Aldrich) were blocked and incubated with primary antibodies at 4 °C overnight followed by labeling with HRP-conjugated secondary antibodies at room temperature for 1 h. The incubated membranes were developed with HRP substrate reagent (Genetex, CA, USA) and recorded with an ECL imaging system ChemiDoc MP (Bio-Rad Laboratories, CA, USA).