Spark instrument

We used commercially available kits to measure serum levels of IGF-1 (see Key Resources Table) according to the manufacturer’s instructions. Measurements were taken using a Tecan SPARK instrument.

Gram staining was performed using a commercial Gram staining kit (Hangzhou Microbiology Reagent Co., Ltd.), and stained bacteria were observed under a light microscope. Bacteria used for scanning electron microscopy (SEM) were cultured in 2216E liquid medium overnight at 28°C and fixed. Briefly, the bacteria were fixed in 2.5% (wt/vol) glutaraldehyde for 3 h and then dehydrated by consecutive washes in ethanol (30%, 50%, 70%, 85%, and 90% ethanol), followed by 2 washes in absolute isopentyl acetate. The fixed bacterial cells were examined on microscope slides using a Hitachi Regulus 8230 scanning electron microscope. Biochemical characterizations were carried out using a commercial kit for Vibrio identification consisting of a panel of test substrates for bacteria to grow on 1% NaCl dissolved lysine, arginine, ornithine, glucose, sucrose, mannitol, and citrate and 6% NaCl dissolved peptone (Vibrio biochemical identification kit; Hangzhou Microbiological Reagent Co., Ltd.). The optimal growth temperature was determined using the 2216E liquid medium in a temperature range of 8°C to 44°C with a step difference of 4°C. The absorbance unit at every 3 h of incubation was measured at an optical density at 600 nm (OD₆₀₀) using a Tecan Spark instrument, which was used to carry out the growth curve. All biochemical tests were performed in triplicate.

In vitro degradation assays containing 1 μM ClpC1 (hexamer), 1 μM ClpP1 (tetradecamer), 1 μM ClpP2 (tetradecamer), and 10 μM substrate were performed in ClpC1 protein degradation buffer. All degradation assays were carried out in the presence of 50 μM activator peptide Z-Leu-Leu-Nva-CHO (benzyloxycarbonyl-l-leucyl-l-leucyl-l-norvalinal) (12 (link), 13 (link)), with a total of 15 mM ATP, along with an ATP regeneration system consisting of 187.5 U mL⁻¹ pyruvate kinase and 50 mM phosphoenolpyruvate (Sigma). For gel degradation assays, 14-μL aliquots were taken at each time point, mixed with 7 μL 2× Laemmli sample buffer (containing 10% β-mercaptoethanol), and analyzed by SDS-PAGE. Gels were stained by 0.1% Coomassie brilliant blue and quantified by ImageJ (88 (link)). Plate reader assays were carried out on a Tecan Spark instrument. Degradation of GFP-substrate fusions was monitored by loss of 511-nm emission following excitation at 450 nm. ATPase assays utilized 1 μM ClpC1, 10 mM ATP, and an NADH-coupled ATP regeneration system (89 (link)). Consumption of ATP was followed by monitoring the decrease in NADH absorbance at 340 nm. Microscale thermophoresis was performed in a Monolith NT.115 (NanoTemper) using 0.1 μM ^H7-SUMOClpC1^FLAG (hexamer) and 0.1 μM His Lite OG488-Tris-NTA-Ni dye (AAT Bioquest). Data were fit to a Hill-form binding equation in Prism (GraphPad).