Amicon ultra 3k

The supernatants from infected HSC‐3 (sh‐NC, sh‐RACK1, OE‐vector or OE‐RACK1) cells cultured in serum‐free DMEM were harvested after 48 h. The infected HSC‐3 cell supernatants were filtered using 0.45‐μm polyvinylidene difluoride membrane filters and concentrated by ultrafiltration (Amicon Ultra 3K; Merck Millipore, Billerica, MA, USA). The Bradford method was employed to determine the protein concentrations. THP‐1 cells (2 × 10⁷ per well) or RAW264.7 cells (1 × 10⁶ per well) were added to the upper compartment of a 24‐well Transwell chamber and then cocultured for 24 h with DMEM containing 40 μg·mL⁻¹ infected OSCC supernatants. The lower compartment contained DMEM supplemented with 2% FBS. The migrated THP‐1 cells were counted by flow cytometry, and the migrated RAW264.7 cells were fixed, stained with Cell Stain Solution (Sigma‐Aldrich, St Louis, MO, USA) and photographed under a light microscope.

C. jejuni were incubated in DMEM (−) overnight and the bacterial cells were removed using a syringe filter (Corning, #431215). After that, the flow through was ultra filtered using an Amicon Ultra 3K device (Merck Millipore, #UFC800324) at 3,000×g for 60 min. The culture medium on HeLa cells were replaced with the ultrafiltered flow through and evaluated autophagy induction.

Extracellular parasites (10⁸) were hypotonically lysed in 1 mL of sterile deionized water containing protease inhibitors (Sigma) by six freeze–thaw cycles (5 min each/liquid nitrogen/37 °C). After concentrating over ten times using centrifugal filter units (Amicon ultra‐3K, Merck, Kenilworth, NJ, USA), samples were centrifuged (30 000 g/30 min/4 °C) 33. The supernatant, containing all soluble proteins, constituted the soluble fraction. The pellet was lysed in 1% SDS (1 h/room temperature) to extract all membrane proteins and centrifuged (30 000 g/30 min/4 °C); 30 μL each of the soluble and pellet (1% SDS supernatant) fractions was resolved by SDS/PAGE and subjected to western blotting as described previously. Here, the PVDF membrane was blocked in 3% BSA‐PBS for 1 h, incubated in primary antibodies [1 : 500 mouse anti‐GFP (Roche) and 1 : 10 000 rabbit anti‐BiP (a kind gift from J. Bangs) 34 for 4 h, and incubated in HRP‐conjugated secondary antibodies for 1.5 h prior to development with diaminobenzidine.

To conjugate AAs with CEs, 15 μL of a 10 mg/mL AA solution (containing either histidine, cysteine, lysine, arginine, glutamine, glutamic acid, tryptophan, proline, or glycine) in phosphate buffer (10 mM, pH = 7.4), was mixed with 10 μL of either 4OHE2 or 2OHE2 (5 mg/mL in acetonitrile), and diluted to 75 μL with the phosphate buffer. Acetonitrile was evaporated from the solution by purging with nitrogen gas until a final volume of ~52 μL was reached. The mixture was incubated at 37 °C for 50 min and then diluted with formic acid (0.1%) to a final volume of 1 mL.
Similar procedures were followed for the insulin conjugation experiments. Briefly, 20 μL of a 4OHE2 solution (5 μg/μL) were added to 100 μL of insulin (Humulin R) or teriparatide (Forteo) solution (1 μg/μL), and the mixtures were incubated at 37 °C for 24 h (or other specified times). Unbound 4OHE2 was removed by centrifugation (16,286 × g) through a 3 kDa cutoff spin column (Amicon Ultra 3 K, Merck Millipore, Darmstadt, Germany). The resulting mixture of insulin conjugation reaction (CE-Ins) composed of un-modified insulin and 4OHE2-conjugated insulin (>30%).

TTX extraction from blood was performed according to previously described procedures [19 (link),42 (link)]. A slightly modified protocol was applied for TTX extraction from feces, urine, and organs. Briefly, mouse blood (100 µL) or plasma (15–53 µL) was mixed with 800 µL of 2% acetic acid and vortexed for 5 min. The mixture was transferred to an ultrafiltration spin column (Amicon^® Ultra 3K centrifugal filters, Merck Millipore, Darmstadt, Germany) and centrifuged at 4000 rpm for 40 min. Mouse feces (0.3 g) were mixed with 1600 µL of 2% acetic acid and vortexed for 5 min, and the extract was centrifuged at 4000 rpm for 15 min. The supernatant was transferred to the ultrafiltration spin column and additionally centrifuged at 4000 rpm for 60 min. Mouse kidney (0.067–0.257g) and mouse liver (0.177–0.297g) were cut into small pieces (<1 mm³), mixed with 800 µL of 2% acetic acid and vortexed for 5 min, then extracted again with 800 µL of the same solution. Supernatants were pooled, filtered through 0.22 µm, transferred to an ultrafiltration spin column, and additionally centrifuged at 4000 rpm for 30 min. In all cases, ultrafiltered solutions were dried, dissolved in 200 µL of 0.03M acetic acid, and filtered again through 0.45 µm filters before placing then in the LC-MS vial for analysis. The same protocol was applied to blood, feces, and tissue samples from control animals.