Anti myc

Total cellular protein (60 μg) was loaded onto a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and electrophoresis was performed, followed by electroblotting to a PVDF membrane (Invitrogen). The blots were incubated with anti-myc (Invitrogen) and anti β-Actin (Invitrogen) antibodies as previously described [26 (link)]. Signals were detected using the luminescent image analyzer ImageQuant LAS 4000 (GE Healthcare, CT, USA).

Protein detection in the cell wall was performed by fixing 4-days-old seedlings in 100% (v/v) methanol for 30 s at room temperature. Epitopes were blocked by incubating seedlings for 2 h with Tris-buffered saline [TBS; 20 mM Tris (pH of 8), 150 mM NaCl] plus 3% bovine serum albumin. Seedlings were incubated overnight with primary antibody 1:1000 anti-myc (Invitrogen, USA) in TBS and 0.5% bovine serum albumin. Washes were performed using phosphate buffered saline plus 1% Triton X-100. Then roots were incubated in secondary antibody anti-mouse-Alexa488 (Invitrogen, USA) diluted 1:1000 in TBS 0.5% BSA. Seedlings were washed using phosphate buffered saline plus 1% Triton X-100 and mounted on 50% glycerol for confocal imaging.
Pectin detection in the cell wall was performed by fixing the cells in 4% paraformaldehyde, 50 mM Pipes, 5 mM MgSO₄, and 5 mM EGTA. Blocking was performed using TBS plus 5% fat free milk. Overnight incubation utilized primary antibody 1:100 Jim5, Jim7, Jim11, Jim14, LM1, LM2, CCRCM1, or CCRCM4 in TBS and 0.5% fat free milk. Then, roots were incubated in secondary antibody anti-rat Alexa 488 diluted 1:1000 in TBS and 0.5% fat free milk. Washes were performed using TBS plus 0.05% Triton X-100. Seedlings were mounted on 50% glycerol for confocal imaging.