Ha 3f10

The antibodies and reagents used in this study were as follows: GFP (A6455; rabbit; Invitrogen; Chen et al., 2011 (link)); MAP2 (AB5622; rabbit; EMD Millipore; Chen et al., 2011 (link)); MAP2 (M4403; mouse; Sigma-Aldrich; Chen et al., 2011 (link)); SMI-312R (SMI-312R; mouse; Covance; Liu et al., 2013 (link)); HA (3F10; rat; Roche; Chen et al., 2017 (link)); phospho-P38 MAPK (9211; rabbit; Cell Signaling Technology); rabbit polyclonal P38 MAPK antibody (9212; rabbit; Cell Signaling Technology); phospho-ERK (4376; rabbit; Cell Signaling Technology); ERK (4695; rabbit; Cell Signaling Technology); phospho-JNK (9251; rabbit; Cell Signaling Technology); JNK (9252; rabbit; Cell Signaling Technology); phospho-TAK1 (9339; rabbit; Cell Signaling Technology); GAPDH (sc-25778; rabbit; Santa Cruz Biotechnology, Inc.; Chen et al., 2011 (link)); NeuN (MAB377; mouse; EMD Millipore; Wang et al., 2015 (link)); HRP-conjugated secondary antibodies (GE Healthcare); and Alexa Fluor 488– and Alexa Fluor 594–conjugated secondary antibodies (Invitrogen). Antibodies with validation profiles in Antibodypedia or 1DegreeBio are underlined. CL075, poly dT, poly(I:C) high molecular weight, and SB203580 were all purchased from InvivoGen. Takinib and NG 25 were purchased from Medchem Express, and (5Z)-7-oxozeaenol was purchased from Tocris.

The paraffin‐embedded tissue sections were deparaffinized in xylene, rehydrated in ethanol, and immersed in citrate‐NaOH buffer (10 mmol/L sodium citrate, pH 6.0) at 121°C for 20 minutes. After retrieval of antigenicity, the nonspecific Ab reaction was blocked in blocking solution (PerkinElmer Life Sciences), and the samples were incubated with Abs against HA (3F10; Roche Diagnostics), E‐cadherin (610181; BD Biosciences), and Ki‐67 (Abcam). After the sections had been washed, the reacted Abs were detected using the Dako EnVision+ System/HRP (DAB) (DakoCytomation).

The protein samples were subjected to SDS‐PAGE. The proteins were then electrotransferred to PVDF membranes (Millipore) and subjected to immunoblot analysis. Antibodies against FLAG (M2; Sigma‐Aldrich), HA (3F10; Roche Diagnostics), c‐Myc (9E10; Santa Cruz Biotechnology), and phosphorylated tyrosine (4G10; Millipore) were used. The reacted Abs were detected as described previously.22

Antibodies against HA (3F10; Roche), p38 MAPK (Cell Signaling Technology), Phospho-p38 MAPK (Cell Signaling Technology), Phospho-c-Fos (Ser32; Cell Signaling Technology), H4 Acetylated (EMD Millipore), EGR1 (Cell Signaling Technology), Phospho-ATF-2 (Thr71; Cell Signaling Technology), and HAUSP (Bethyl Laboratories) were used in the immunofluorescence assay and/or in western blotting. Immunofluorescence secondary antibodies were coupled with Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen). Secondary antibodies used in western blotting were conjugated to alkaline phosphatase (Promega). The p38 inhibitors were purchased from InvivoGen and Euromedex.

Yeast cell pellets (∼1.5–6.0 OD units) were suspended in 200 µl of 20% trichloroacetic acid, kept on ice for 15 min, and centrifuged at 15,000 g for 5 min. The pellets were washed with 1 ml of cold acetone, dried at room temperature, and suspended in (OD units × 50) µl of SDS sample buffer by mixing at 65°C for 10 min followed by cell disruption at room temperature using FastPrep-24 (MP Biomedicals) and 0.5-mm YZB zirconia beads (Yasui Kikai). These samples were boiled for 3 min and centrifuged at 15,000 g for 1 min; the supernatants were used for immunoblotting analysis (Nakatogawa and Ohsumi, 2012 (link)). To separate phosphorylated and unphosphorylated forms of nontagged Atg19, the Anderson gel system (10% acrylamide and bis-acrylamide at 77:1) was used (Anderson et al., 1973 (link)). Monoclonal antibodies against GFP (11814460001; Roche), AID (BioROIS), GST (B-14; Santa Cruz Biotechnology, Inc.), HA (3F10; Roche), and Myc (9E10; Santa Cruz Biotechnology, Inc.) sequences were used for detection of tagged proteins. The monoclonal antibody against Pgk1 was purchased from Invitrogen. The phosphate-binding reagent Biotinylated Phos-tag (Wako Pure Chemical Industries) was used to detect protein phosphorylation in combination with streptavidin-HRP (Invitrogen).