Aprotinin

We used anti-CD3 and -CD28 monoclonal antibodies (555336, 555725, 553058, 553295; BD PharMingen) for T cell stimulation. For cytometry analysis, we used anti-human-CD69-PE, anti-mouse CD44-FITC (IM1943, 731957; Beckman Coulter), CD4-PECy5, CD8-PeCy7 (100434, 100722; Biolegend), CD69-FITC, CD71-PE (553236, 553267; Pharmingen) and the isotype control mouse IgG1-PE (556029; Pharmingen). For western blot, we used anti-pERK 1/2 (T202/Y204), -ERK 1/2, -pPKD S744/748, -pAKT T473, -Akt, -IκB, -pS6K (T389), -S6K, -prpS6 (S235/236), -pPan-PKC substrate (4370, 4696 S, 2054 L, 4060, 2910 S, 9242 S, 9206 L, 2708, 2211 S, 2261 L; Cell Signaling), -PKD, -GAPDH (sc-935, sc25778; Santa Cruz), anti-α-tubulin (9026; Sigma-Aldrich), -DGKζ, -SNX27, -GLUT1 (αβ105195, ab77799, ab15309; Abcam), anti-Kidins220 described in^{43 (link)} was a kind gift from Dr Teresa Iglesias. The following secondary antibodies were used: horseradish peroxidase (HRP)-conjugated anti-mouse and -rabbit IgG (P0447, P0448; Dako), anti-rabbit IgG Dylight 800 (SA5-35571; Thermo Scientific), AlexaFluor 680-anti-mouse IgG (A-21057; Life Technologies).
Leupeptin and aprotinin were purchased from Roche. We used Na₃VO₄, PMSF, β-glycerophosphate, paraformaldehyde (PFA), cycloheximide (CHX), concanavalin A (ConA), BSA and NP40 (all from Sigma-Aldrich). Gö6976, PD98059 and MG-132 were from Calbiochem.

Alginic acid sodium salt (10 mg/ml, Sigma, USA),
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
(HEPES, 25 mM, Sigma, USA), and sodium chloride (NaCl,
150 mM, Sigma, USA) were dissolved in deionized water
to make a 1.0% (w/v) ALG solution (20 (link)). Immediately
before use, the sterilized ALG solution was reconstituted
with sterile 1X phosphate-buffered saline (PBS, Takara, Japan) without calcium and magnesium to yield a 0.5%
(w/v) concentration. FA solution was prepared by mixing
fibrinogen solution [50 mg/ml fibrinogen (Sigma, USA)
in 3000 KIU/mL aprotinin (Roche, Germany)] with 1%
ALG solution at 1:1 ratio. HAA solution was made by the
addition of HA [5 mg/ml, Nano Zist Arrayeh (NZA), Iran]
to a 0.5% ALG solution (8 (link), 20 (link)).
In order to prepare the hydrogels, cross-linking solutions [50 mM calcium chloride
(CaCl₂, Sigma, USA)/140 mM NaCl for making ALG and HAA, and 50 mM
CaCl₂/140 mM NaCl with the equal volume of 50 IU/ml thrombin solution for FA]
were mixed with the hydrogel solutions.

The metalloprotease inhibitor, 1,10-Phenanthroline (Invitrogen), was prepared as a 2 M stock solution in ethanol. The cysteine protease inhibitor iodoacetamide (G-Biosciences, St. Louis, MO, or Sigma-Aldrich) was prepared as a 10 mM stock solution in HyPure H₂O (Cytiva Life Sciences, Marlborough, MA). The serine protease inhibitor aprotinin (Roche, Mississauga, ON) was prepared as a 0.1 mM stock solution in HyPure H₂O. Working solutions of protease inhibitors were diluted in EnzChek reaction buffer (Invitrogen) or TBS and incorporated into collagenase, caseinase, and fibrinogen degradation assays at the concentrations reported in the figure legends.

Cell extracts were prepared in RIPA buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitors (1 mM Na₂VO₄, 10 mM NaF, 2 mM PMSF, 5 μg/ml Leupeptin, 10 μg/ml Aprotinin, 1 μg/ml Pepstatin A) (Roche, Switzerland). Equal amounts of proteins were separated by SDS-PAGE followed by electrotransfer onto PVDF membranes (PALL Life Sciences, USA), Membranes were subsequently incubated with appropriate primary antibodies overnight at 4 °C, followed by incubation with peroxidase-conjugated secondary antibodies for 1 h at room temperature. The bands were visualized by using the ECL chemiluminescent detection system (iNtRON Biotechnology, South Korea). For immunoprecipitation of protein complexes, cell extracts were pre-cleared with protein G-Sepharose beads (GE Healthcare, USA) and incubated with appropriate antibodies. Immune complexes were then analyzed by immunoblotting.