Figure EV3. TPL‐2 regulates phagosome proteolysis via DMXLI.

• A
  Dmxl1 was knocked down in WT iBMDMs by RNA interference using a SMARTpool ON‐TARGETplus siRNA for 48 h. ON‐TARGETplus non‐targeting pool functioned as siRNA control. Intra‐phagosomal proteolysis was assayed following uptake of DQ Green BSA / AF594 latex beads (n = 4 wells).
• B–D
  Dmxl2 was knocked down in WT iBMDMs by RNA interference using a SMARTpool ON‐TARGETplus siRNA for 48 h. ON‐TARGETplus non‐targeting pool functioned as siRNA control. (B) Intra‐phagosomal proteolysis was assayed following uptake of DQ Green BSA / AF594 latex beads (n = 4 wells). (C) Intra‐phagosomal acidification was assayed following uptake of BCECF‐coupled latex beads (n = 4 wells). (D) qRT–PCR analysis of RNA extracted from iBMDMs was used to check the efficiency of Dmxl2 knockdown (A and B). Dmxl2 mRNA levels were normalised to Hprt mRNA levels and fold changes calculated (ΔC_t values) (n = 4).
• E
  Dmxl1 was knocked down in Tpl2[D270A] iBMDMs by RNA interference using a SMARTpool ON‐TARGETplus siRNA for 48 h. ON‐TARGETplus non‐targeting pool functioned as siRNA control. Intra‐phagosomal proteolysis was assayed following uptake of DQ Green BSA / AF594 latex beads (n = 4 wells).
• F
  Simultaneous with Dmxl1 siRNA knockdown, WT iBMDMs were co‐transfected with plasmids expressing either 3xFLAG‐DMXL1 (1,773–2,047) or 3xFLAG‐DMXL1 S1903A/S1904A (1,773–2,047). Intra‐phagosomal proteolysis was assayed following uptake of DQ Green BSA / AF594 latex beads (n = 4 wells).
• G
  Simultaneous with Dmxl1 siRNA knockdown, Tpl2[D270A] iBMDMs were co‐transfected with a plasmid expressing 3xFLAG‐DMXL1 (1,773–2,047). Intra‐phagosomal proteolysis was monitored (n = 4 wells).

Data information: (A‐G) One representative experiment out of three shown. Error bars and shaded areas represent SEM. ****P < 0.0001. Paired Mann–Whitney t‐test. All differences relative to WT are ****. UT, untransfected; NT, non‐targeting siRNA pool.