Figure 4. GFPT is a substrate of TG2 transamidase activity.

• A
  HeLa cells were infected with C. trachomatis (MOI = 1), and 40 μM CP4d was added or not 2 hpi. After 24 h, 0.5 mM BP was added and cells were lysed at 48 hpi. Lysates were precipitated with streptavidin‐coated beads. After separation with SDS–PAGE, proteins were transferred to a membrane and blotted with anti‐GFPT antibody followed with HRP‐conjugated secondary antibody.
• B
  In vitro assay testing the ability of purified TG2 to cross‐link purified rhGFPT1 with BP. Samples were incubated for 3 h at 37°C before separation by SDS–PAGE. Proteins were transferred to a membrane, and BP was revealed using HRP‐conjugated streptavidin. rhGFPT1 is 77.5 kDa.
• C
  GFPT1 sequence: Glutamine residues identified by mass spectrometry as cross‐linked to BP are in bold letter.
• D
  In vitro assay was performed as described in (B) using wild‐type rhGFPT1 (WT), rhGFPT1 Q58N, rhGFPT1 Q328N, or rhGFPT1 Q555N as substrates. The reaction was performed at 37°C for 30 min. After probing with HRP–streptavidin, the membrane was washed and probed with anti‐GFPT antibodies followed with HRP‐conjugated secondary antibodies. The ratio of modified protein (streptavidin signal) to the total GFPT is shown and normalized to its value with WT rhGFPT1. The mean ± SD of five independent experiments is shown, and the P‐value of the Student's ratio‐paired t‐test is indicated.
• E
  Lysates of cells treated or not for 6 h with ionomycin were incubated at 37°C for 45 min with fructose‐6‐P and glutamine. The production of glucosamine‐6‐P was measured using HPAEC‐PAD. Results of three independent experiments are shown, with mean ± SD, and P‐value of the Student's paired t‐test is indicated (*P < 0.05).

Source data are available online for this figure.