Iodacetamide

Cells were biotin labelled as described previously after serum starvation in serum free DMEM. Cells were washed in PBS, transferred to serum free DMEM pre-warmed to 37°C and incubated at 37°C in previously described conditions for a 30 minute internalisation period. Post-internalisation, all but 2 wells underwent biotin removal from cell surface proteins by the previously described reduction step. These wells were then quenched with 20 mM iodacetamide (Sigma) (dissolved in PBS) at 4°C for 30 minutes. Two of these wells were lysed as described previously to enable us to quantify the intracellular biotinylated occludin pool after 30 minutes internalisation. In the two remaining non-reduced wells, cells were quenched as before then lysed to measure total biotinylated occludin (plasma membrane and intracellular biotinylated occludin). The remaining dishes were incubated at 37°C in the previously described conditions for 5, 15, 30 or 60 minutes. After each time-point two dishes were reduced and quenched (to measure the intracellular biotinylated occludin pool) while two dishes were quenched (to measure the total biotinylated occludin pool). Dishes were lysed, the biotinylated proteins were pulled down with NeutrAvidin beads and the proteins eluted from the beads and prepared as described previously.

All chemicals were of analytical grade, if not otherwise stated. Sodium chloride (NaCl), sodium hydroxide (NaOH), SDS, 2‐(N‐moprholino)ethanesulfate acid (MES), Tween‐20, sulfuric acid (95–97%, H₂SO₄), uranyl acetate were purchased from Merck (Darmstadt, Germany).
4‐(2‐Hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) (≥99.5%), 2‐propanol, bovine serum albumin (BSA) (≥99.5%), 1,4‐dithiotreitol (DTT), anti‐mouse IgG (γ‐chain specific)‐alkaline phosphatase antibody (#3438), BCIP^®/NBT solution, triton X‐100, glutaraldehyde solution (grade I), acetonitrile (MS grade), formic acid (98–100%), and iodacetamide (≥99%) were purchased from Sigma Aldrich (St. Louis, MO, USA). Anti‐rabbit IgG (H+L) secondary antibody (#31460), anti‐mouse IgG (H+L) superclonal secondary antibody (#A28177) were purchased from Thermo Fisher (Waltham, MA, USA). SeeBlue^® plus 2 prestained protein standard and 4× LDS sample buffer were purchased from Invitrogen (Carlsbad, CA, USA). C‐LEcta Denarase^® was purchased from VWR (Radnor, PA, USA), HIV‐1 p24 antibody (ab9071) and ACV5 (ab49581) from Abcam (Cambridge, UK), influenza A virus H1N1 HA (GTX127357) from GeneTex (Irvine, CA, USA) and trypsin from Promega (Madison, WI, USA).

EBP samples were incubated in 2% sodium dodecyl sulfate (SDS, Sigma-Aldrich, St. Louis, USA) in 50 mM Triethylammonium bicarbonate (TEAB, Thermo Fisher Scientific) at 37 °C for 2 h with subsequent addition of 400 mM dithiothreitol (Sigma-Aldrich) and further incubation for 45 min.. Alkylation was performed in the dark for 30 min with the addition of 800 mM iodacetamide (Sigma-Aldrich) after which 12% aqueous phosphoric acid was added to a final concentration of 1.2%. Proteins were collected onto S-TRAP columns (Protifi, Farmingdale, USA) with a mixture of 90% methanol and 100 mM TEAB. Digestion of proteins was performed with 1 µg of Lys-C (Lys-C, Mass Spec Grade, Promega, Fitchburg, USA) incubated at 37 °C for 2 h after which 1 µg of trypsin (Promega sequence grade) was added overnight with addition of 0.45 µg trypsin after 12 h. Peptides were then eluted with 50 mM TEAB, 0.2% formic acid (FA, Sigma-Aldrich) and 50% acetonitrile (ACN, Sigma-Aldrich) with 0.2% formic acid and dried by speedvac (Eppendorf, Hamburg, Germany) at 45 °C and re-dissolved in 20 uL of 0.1% FA and 2% ACN solution.

0.1 mg from each tissue specimen were cut into small pieces, mixed with lysis buffer (200 mM HEPES, pH 7.5 (AppliChem, Germany), 1% acid-labile surfactant (ALS, sodium 3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl) methoxy]-1-propanesulfonate)), and incubated at 90 °C for 10 min with mild agitation (500 rpm). Subsequently, samples were further homogenized using a PreCellys Lysis Kit (Bertin instruments, USA) for three cycles (each 30 secundum, at 6500 rpm) and sonicated with a Biorupture device for 20 cycles (each 30 secundum ON and 30 secundum OFF). After centrifugation (at 4 °C, for 10 min at max speed), the supernatant was collected, the pH value was adjusted to pH 8.0 and samples were reduced (5 mM TCEP (Sigma Aldrich, Switzerland), 10 min at 95 °C) and alkylated (10 mM iodacetamide (Sigma Aldrich, USA) in darkness at room temperature for 30 min). A double digest with Lysyl Endopeptidase (Wako, Japan) in a ratio of 1:100 for 2 h, at 37 °C, at 500 rpm and trypsin TPCK treated (Worthington, USA) in a ratio of 1:50 overnight at 37 °C, at 500 rpm was performed.