gapdh loading control  (Proteintech)

Journal: mBio

Article Title: A bacterial family of fatty acid acyltransferases related to the Shigella effector IcsB

doi: 10.1128/mbio.03890-25

Figure Lengend Snippet: A subset of novel k-FATs exhibits yeast toxicity and IcsB-like fatty acid acyltransferase activity. ( A ) Map of the pRS313 Gal1p2 centromeric plasmid used for the galactose-inducible expression of IcsB and its homologs. ( B )The expression of IcsB and seven of its homologs in the presence of galactose inhibits yeast growth compared to the empty vector (EV) control. The glucose plate indicates that there is no growth inhibition in the absence of expression and that similar amounts of each strain were plated. Four dilutions were plated (non-diluted, 1/10, 1/100, and 1/1,000). The graph represents the quantification of growth on three independent replicates of the 1/1,000 dilution. The error bars represent the standard deviation of the mean. A Student’s t-test with unpaired data and a 95% CI was performed (*, P < 0.05; **, P < 0.1, ***, P < 0.01). ( C ) Protein acylation in the membrane protein fraction of yeast expressing IcsB and its homologs according to the in-gel fluorescence acyltransferase assay (upper panels). -Alk IcsB is the IcsB sample prior to the addition of Alk-16. -Alk IcsB, Alk-16 EV, and Alk-16 IcsB were loaded on both gels to serve as controls. This acylation assay was performed using the strains from B that were induced with galactose for six hours in the presence of Alk-16 for the final 4 h. The total protein in these samples were assessed using the TGX stain (bottom panels). ( D ) The expression of IcsB and of its homologs was assessed by immunoblotting with an antibody binding to their 3× FLAG tag and with the GAPDH as a loading control.

Article Snippet: Primary antibodies used were mouse FLAG-tag (Sigma-Aldrich, F3165), mouse Myc-tag (Genescript, A00704), and rabbit GAPDH (Bioss antibodies, BS-8789R) diluted 1/5,000, 1/1,000, and 1/10,000, respectively.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Control, Inhibition, Standard Deviation, Membrane, Fluorescence, Staining, Western Blot, Binding Assay, FLAG-tag

Journal: mBio

Article Title: A bacterial family of fatty acid acyltransferases related to the Shigella effector IcsB

doi: 10.1128/mbio.03890-25

Figure Lengend Snippet: Mutations in the catalytic site of k-FATs abolish their enzymatic activity. ( A ) Yeast growth inhibition tests of catalytic residues mutants H145A (left), D195A (center), and C306A (right) compared to their WT counterpart in IcsB and in toxic homologs. The graph represents the quantification of growth on three independent replicates with the dilution of yeast cultures 1/1,000. The error bars represent the standard deviation from the mean. A Student’s t -test with unpaired data and a 95% CI was performed (*, P < 0.05; **, P < 0.1, ***, P < 0.01). ( B ) In-gel protein acylation of C306A mutants compared to their WT counterparts (upper panels). The total protein in these samples were assessed using the TGX stain (bottom panels). ( C ) The expression of IcsB and toxic homolog C306A mutants was compared to their WT counterparts by immunoblotting with an antibody binding to their 3×FLAG tag and with the GAPDH as a loading control.

Article Snippet: Primary antibodies used were mouse FLAG-tag (Sigma-Aldrich, F3165), mouse Myc-tag (Genescript, A00704), and rabbit GAPDH (Bioss antibodies, BS-8789R) diluted 1/5,000, 1/1,000, and 1/10,000, respectively.

Techniques: Activity Assay, Inhibition, Standard Deviation, Staining, Expressing, Western Blot, Binding Assay, Control